Weed controller metabolism proteins, genes thereof and use of the same

ABSTRACT

The present invention provides, for example, DNA encoding a herbicide metabolizing protein selected from the protein group below. Such DNA may, for example, be employed to produce herbicidally resistant plants. &lt;protein group&gt;a protein comprising the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 108, 159, 160, 136, 137, 138, 215, 216, 217, 218, 219, 220, 221, 222, 223 or 224, 
 
a protein having an ability to convert in the presence of an electron transport system containing an electron donor, a compound of formula (II):  
                 
 
to a compound of formula (III):  
                 
and comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence shown in any one of SEQ ID NO: 1, 2, 3, 108, 159, 136, 137, 138, 215, 216, 217, 218, 219, 220, 221, 222, 223 or 224 or an amino acid sequence having at least 90% sequence identity with an amino acid sequence shown in any one of SEQ ID NO: 160, 215, 216, 218, 222 or 224.

TECHNICAL FIELD

The present invention relates to a protein having the ability tometabolize a herbicidal compound (Herbicide metabolizing protein), agene thereof and use thereof.

BACKGROUND ART

Herbicides are utilized in a necessary amount of diluted solution whenapplied. There are situations in which extra amounts are left over.There are also situations in which the applied herbicide, after itsapplication for awhile, remains in the soil or plant, residue.Originally, given that the safety of such herbicides has been checked,such small amounts of left-over solutions or residues presented smalleffects to the environment or to the crops cultivated thereafter.However, if there is a method in which the contained herbicidal compoundis converted to one of lower herbicidal activity, then for example therecan be conducted treatments to inactivate the left-over solutions orresidues described above as needed.

Further, in the case of using the herbicide, there were situations inwhich it was difficult to distinguish cultivated plants from weeds ofallied species to selectively control only weeds. Then, there is adesire to develop a new method for conferring herbicidal resistance to atarget plant.

DISCLOSURE OF THE INVENTION

Under such the circumstances, the present inventors intensively studiedand, as a result have found that a protoporphyrinogen oxidase(hereinafter, sometimes referred to as “PPO”) inhibitory-type herbicidalcompound may be converted by being reacted with a particular protein toa compound of lower herbicidal activity, which resulted in completion ofthe present invention.

That is, the present invention provides:

-   1. A DNA encoding a herbicide metabolizing protein, wherein said    protein is selected from the group consisting of:-   (A1) a protein comprising the amino acid sequence shown in SEQ ID    NO: 1;-   (A2) a protein comprising the amino acid sequence shown in SEQ ID    NO: 2;-   (A3) a protein comprising the amino acid sequence shown in SEQ ID    NO: 3;-   (A4) a protein comprising the amino acid sequence shown in SEQ ID    NO: 108;-   (A5) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II):    -   to a compound of formula (III):    -   and comprising an amino acid sequence having at least 80%        sequence identity with an amino acid sequence shown in any one        of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 108;-   (A6) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    80% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID    NO: 3 or SEQ ID NO: 108;-   (A11) a protein comprising the amino acid sequence shown in SEQ ID    NO; 159;-   (A12) a protein comprising the amino acid sequence shown in SEQ ID    NO; 160;-   (A13) a protein comprising the amino acid sequence shown in SEQ D    NO: 136;-   (A14) a protein comprising the amino acid sequence shown in SEQ ID    NO: 137;-   (A15) a protein comprising the amino acid sequence shown in SEQ ID    NO: 138;-   (A16) a protein comprising the amino acid sequence shown in SEQ ID    NO: 215;-   (A17) a protein comprising the amino acid sequence shown in SEQ ID    NO: 216;-   (A18) a protein comprising the amino acid sequence shown in SEQ ID    NO: 217;-   (A19) a protein comprising the amino acid sequence shown in SEQ ID    NO; 218;-   (A20) a protein comprising the amino acid sequence shown in SEQ ID    NO: 219;-   (A21) a protein comprising the amino acid sequence shown in SEQ ID    NO: 220;-   (A22) a protein comprising the amino acid sequence shown in SEQ ID    NO: 221;-   (A23) a protein comprising the amino acid sequence shown in SEQ ID    NO: 222;-   (A24) a protein comprising the amino acid sequence shown in SEQ ID    NO: 223;-   (A25) a protein comprising the amino acid sequence shown in SEQ ID    NO: 224;-   (A26) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO:    136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219,    SEQ ID NO: 220, SEQ ID NO: 221 or SEQ ID NO: 223 or an amino acid    sequence having at least 90% sequence identity with an amino acid    sequence shown in any one of SEQ ID NO: 160, SEQ ID NO: 215, SEQ ID    NO: 216, SEQ ID NO: 218, SEQ ID NO: 222 or SEQ ID NO: 224;-   (A27) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    90% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO; 160,    SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 215, SEQ    ID NO, 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO: 219, SEQ ID    NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 or SEQ ID    NO: 224; and-   (A28) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a DNA amplifiable by a polymerase    chain reaction with a primer comprising the nucleotide sequence    shown in any one of SEQ ID NOs: 124 to 128, a primer comprising the    nucleotide sequence shown in SEQ ID NO: 129 and as a template a    chromosomal DNA of Streptomyces phaeochromogenes, Streptomyces    testaceus, Streptomyces achromogenes, Streptomyces griseofuscus,    Streptomyces thermocoerulescens, Streptomyces nogalater,    Streptomyces tsusimaensis, Streptomyces glomerochromogenes,    Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces    griseus, Streptomyces lanatus, Streptomyces misawanensis,    Streptomyces pallidus, Streptomyces roseorubens, Streptomyces    rutgersensis, Streptomyces steffisburgensis or Saccharopolyspora    taberi;-   2. A DNA comprising a nucleotide sequence selected from the group    consisting of:-   (a1) the nucleotide sequence shown in SEQ ID NO: 6;-   (a2) the nucleotide sequence shown in SEQ ID NO: 7;-   (a3) the nucleotide sequence shown in SEQ ID NO: 8;-   (a4) the nucleotide sequence shown in SEQ ID NO: 109;-   (a5) the nucleotide sequence shown in SEQ ID NO: 139;-   (a6) the nucleotide sequence shown in SEQ ID NO: 140;-   (a7) the nucleotide sequence shown in SEQ ID NO: 141;-   (a8) the nucleotide sequence shown in SEQ ID NO: 142;-   (a9) the nucleotide sequence shown in SEQ ID NO: 143;-   (a10) the nucleotide sequence shown in SEQ ID NO: 225;-   (a11) the nucleotide sequence shown in SEQ ID NO: 226;-   (a12) the nucleotide sequence shown in SEQ ID NO: 227;-   (a13) the nucleotide sequence shown in SEQ ID NO: 228;-   (a14) the nucleotide sequence shown in SEQ ID NO: 229;-   (a15) the nucleotide sequence shown in SEQ ID NO: 230;-   (a16) the nucleotide sequence shown in SEQ ID NO: 231;-   (a17) the nucleotide sequence shown in SEQ ID NO: 232;-   (a17) the nucleotide sequence shown in SEQ ID NO: 233;-   (a19) the nucleotide sequence shown in SEQ ID NO: 234;-   (a20) a nucleotide sequence encoding an amino acid sequence of a    protein having an ability to convert in the presence of an electron    transport system containing an electron donor, a compound of    formula (II) to a compound of formula (III), said nucleotide    sequence having at least 80% sequence identity with a nucleotide    sequence shown in any one of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:    8 or SEQ ID NO: 109; and-   (a21) a nucleotide sequence encoding an amino acid sequence of a    protein having an ability to convert in the presence of an electron    transport system containing an electron donor, a compound of    formula (II) to a compound of formula (III), said nucleotide    sequence having at least 90% sequence identity with a nucleotide    sequence shown in any one of SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID    NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 225, SEQ ID NO:    226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230,    SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233 or SEQ ID NO: 234;-   3. The DNA according to the above 1, comprising a nucleotide    sequence encoding an amino acid sequence of said protein, wherein    the codon usage in said nucleotide sequence is within the range of    plus or minus 4% of the codon usage in genes from the species of a    host cell to which the DNA is introduced and the GC content of said    nucleotide sequence is at least 40% and at most 60%;-   4. A DNA comprising the nucleotide sequence shown in SEQ ID NO: 214;-   5. A DNA comprising the nucleotide sequence shown in SEQ ID NO: 368;-   6. A DNA comprising the nucleotide sequence shown in SEQ ID NO: 393;-   7. A DNA in which a DNA having a nucleotide sequence encoding an    intracellular organelle transit signal sequence is linked upstream    of the DNA according to the above 1 in frame;-   8. A DNA in which the DNA according to the above 1 and a promoter    functional in a host cell are operably linked;-   9. A vector comprising the DNA according to the above 1;-   10. A method of producing a vector comprising a step of inserting    the DNA according to the above 1 into a vector replicable in a host    cell;-   11. A transformant in which the DNA according to the above 1 is    introduced into a host cell;-   12. The transformant according to the above 11, wherein the host    cell is a microorganism cell or a plant cell;-   13. A method of producing a transformant comprising a step of    introducing into a host cell, the DNA according to the above 1;-   14. A method of producing a protein having the ability to convert a    compound of formula (II) to a compound of formula (III), said method    comprising a steps of culturing the transformant according to the    above 11 and recovering the produced said protein;-   15. Use of the DNA according to the above 1 for producing a protein    having the ability to convert a compound of formula (II) to a    compound of formula (III);-   16. A method of giving a plant resistance to a herbicide, said    method comprising a step of introducing into and expressing in a    plant cell, the DNA according to the above 1;-   17. A polynucleotide having a partial nucleotide sequence of a DNA    according to the above 1 or a nucleotide sequence complimentary to    said partial nucleotide sequence;-   18. A method of detecting a DNA encoding a protein having the    ability to convert a compound of formula (II) to a compound of    formula (III), said method comprising a step of detecting a DNA to    which a probe is hybridized in a hybridization using as the probe    the DNA according to the above 1 or the polynucleotide according to    the above 17;-   19. A method of detecting a DNA encoding a protein having the    ability to convert a compound of formula (II) to a compound of    formula (II), said method comprising a step of detecting a DNA    amplified in a polymerase chain reaction with the polynucleotide    according to the above 17 as a primer;-   20. The method according to the above 19, wherein at least one of    the primers is selected from the group consisting of a    polynucleotide comprising the nucleotide sequence shown in any one    of SEQ ID NOs: 124 to 128 and a polynucleotide comprising the    nucleotide sequence shown in SEQ ID NO: 129;-   21. A method of obtaining a DNA encoding a protein having the    ability to convert a compound of formula (II) to a compound of    formula (III), said method comprising a step of recovering the DNA    detected by the method according to the above 18 or 19.-   22. A method of screening a cell having a DNA encoding a protein    having the ability to convert a compound of formula (II) to a    compound of formula (III), said method comprising a step of    detecting said DNA from a test cell by the method according to the    above 18 or 19;-   23. A herbicide metabolizing protein selected from the group    consisting of:-   (A1) a protein comprising the amino acid sequence shown in SEQ ID    NO: 1;-   (A2) a protein comprising the amino acid sequence shown in SEQ ID    NO: 2;-   (A3) a protein comprising the amino acid sequence shown in SEQ ID    NO: 3;-   (A4) a protein comprising the amino acid sequence shown in SEQ ID    NO: 108;-   (A5) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III) and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2,    SEQ ID NO: 3 or SEQ ID NO: 108;-   (A6) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    80% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID    NO:3 or SEQ ID NO: 108;-   (A11) a protein comprising the amino acid sequence shown in SEQ ID    NO: 159;-   (A12) a protein comprising the amino acid sequence shown in SEQ ID    NO: 160;-   (A13) a protein comprising the amino acid sequence shown in SEQ ID    NO: 136;-   (A14) a protein comprising the amino acid sequence shown in SEQ ID    NO: 137;-   (A15) a protein comprising the amino acid sequence shown in SEQ ID    NO: 138;-   (A16) a protein comprising the amino acid sequence shown in SEQ ID    NO: 215;-   (A17) a protein comprising the amino acid sequence shown in SEQ ID    NO: 216;-   (A18) a protein comprising the amino acid sequence shown in SEQ ID    NO: 217;-   (A19) a protein comprising the amino acid sequence shown in SEQ ID    NO: 218;-   (A20) a protein comprising the amino acid sequence shown in SEQ ID    NO: 219;-   (A21) a protein comprising the amino acid sequence shown in SEQ ID    NO: 220;-   (A22) a protein comprising the amino acid sequence shown in SEQ ID    NO: 221;-   (A23) a protein comprising the amino acid sequence shown in SEQ ID    NO: 222;-   (A24) a protein comprising the amino acid sequence shown in SEQ ID    NO: 223;-   (A25) a protein comprising the amino acid sequence shown in SEQ ID    NO: 224;-   (A26) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO:    136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 211, SEQ ID NO: 219,    SEQ ID NO: 220, SEQ ID NO: 221 or SEQ ID NO; 223 or an amino acid    sequence having at least 90% sequence identity with an amino acid    sequence shown in any one of SEQ ID NO: 160, SEQ ID NO: 215, SEQ ID    NO: 216, SEQ ID NO: 218, SEQ ID NO: 222 or SEQ ID NO: 224;-   (A27) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    90% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 160,    SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO, 138, SEQ ID NO: 215, SEQ    ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO: 219, SEQ ID    NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO, 223 or SEQ ID    NO: 224; and-   (A28) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a DNA amplifiable by a polymerase    chain reaction with a primer comprising the nucleotide sequence    shown in any one of SEQ ID NOs: 124 to 128, a primer comprising the    nucleotide sequence shown in SEQ ID NO: 129 and as a template a    chromosomal DNA of Streptomyces phaeochromogenes, Streptomyces    testaceus, Streptomyces achromogenes, Streptomyces griseofuscus,    Streptomyces thermocoerulescens, Streptomyces nogalater,    Streptomyces tsusimaensis, Streptomyces glomerochromogenes,    Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces    griseus, Streptomyces lanatus, Streptomyces misawanensis,    Streptomyces pallidus, Streptomyces roseorubens, Streptomyces    rutgersensis, Streptomyces steffisburgensis or Saccharopolyspora    taberi;-   24. An antibody recognizing a herbicide metabolizing protein    selected from the group consisting of:-   (A1) a protein comprising the amino acid sequence shown in SEQ ID    NO: 1;-   (A2) a protein comprising the amino acid sequence shown in SEQ ID    NO: 2;-   (A3) a protein comprising the amino acid sequence shown in SEQ ID    NO: 3;-   (A4) a protein comprising the amino acid sequence shown in SEQ ID    NO: 108;-   (A5) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III) and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2,    SEQ ID NO: 3 or SEQ ID NO: 108;-   (A6) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    80% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID    NO; 3 or SEQ ID NO, 108;-   (A11) a protein comprising the amino acid sequence shown in SEQ ID    NO: 159;-   (A12) a protein comprising the amino acid sequence shown in SEQ ID    NO: 160;-   (A13) a protein comprising the amino acid sequence shown in SEQ ID    NO: 136;-   (A14) a protein comprising the amino acid sequence shown in SEQ ID    NO: 137;-   (A15) a protein comprising the amino acid sequence shown in SEQ ID    NO; 138;-   (A16) a protein comprising the amino acid sequence shown in SEQ ID    NO: 215;-   (A17) a protein comprising the amino acid sequence shown in SEQ ID    NO; 216;-   (A18) a protein comprising the amino acid sequence shown in SEQ ID    NO: 217;-   (A19) a protein comprising the amino acid sequence shown in SEQ ID    NO: 218;-   (A20) a protein comprising the amino acid sequence shown in SEQ ID    NO: 219;-   (A21) a protein comprising the amino acid sequence shown in SEQ ID    NO: 220;-   (A22) a protein comprising the amino acid sequence shown in SEQ ID    NO: 221;-   (A23) a protein comprising the amino acid sequence shown in SEQ ID    NO: 222;-   (A24) a protein comprising the amino acid sequence shown in SEQ ID    NO: 223;-   (A25) a protein comprising the amino acid sequence shown in SEQ ID    NO: 224;-   (A26) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO:    136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219,    SEQ ID NO: 220, SEQ ID NO: 221 or SEQ ID NO; 223 or an amino acid    sequence having at least 90% sequence identity with an amino acid    sequence shown in any one of SEQ ID NO: 160, SEQ ID NO 215, SEQ ID    NO: 216, SEQ ID NO: 218, SEQ ID NO: 222 or SEQ ID NO: 224;-   (A27) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    90% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 160,    SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO, 215, SEQ    ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO: 219, SEQ ID    NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 or SEQ ID    NO: 224; and-   (A28) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a DNA amplifiable by a polymerase    chain reaction with a primer comprising the nucleotide sequence    shown in any one of SEQ ID NOs: 124 to 128, a primer comprising the    nucleotide sequence shown in SEQ ID NO: 129 and as a template a    chromosomal DNA of Streptomyces phaeochromogenes, Streptomyces    testaceus, Streptomyces achromogenes, Streptomyces griseofuscus,    Streptomyces thermocoerulescens, Streptomyces nogalater,    Streptomyces tsusimaensis, Streptomyces glomerochromogenes,    Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces    griseus, Streptomyces lanatus, Streptomyces misawanensis,    Streptomyces pallidus, Streptomyces roseorubens, Streptomyces    rutgersensis, Streptomyces steffisburgensis or Saccharopolyspora    taberi;-   25. A method of detecting a herbicide metabolizing protein, said    method comprising:    -   (1) a step of contacting a test substance with an antibody        recognizing said protein and    -   (2) a step of detecting a complex of said protein and said        antibody, arising from said contact,-    wherein said protein is selected from the group consisting of:-   (A1) a protein comprising the amino acid sequence shown in SEQ ID    NO: 1;-   (A2) a protein comprising the amino acid sequence shown in SEQ ID    NO: 2;-   (A3) a protein comprising the amino acid sequence shown in SEQ ID    NO: 3;-   (A4) a protein comprising the amino acid sequence shown in SEQ ID    NO: 108;-   (A5) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III) and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2,    SEQ ID NO: 3 or SEQ ID NO: 108;-   (A6) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    80% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID    NO: 3 or SEQ ID NO: 108;-   (A11) a protein comprising the amino acid sequence shown in SEQ ID    NO; 159;-   (A12) a protein comprising the amino acid sequence shown in SEQ ID    NO: 160;-   (A13) a protein comprising the amino acid sequence shown in SEQ ID    NO: 136;-   (A14) a protein comprising the amino acid sequence shown in SEQ ID    NO: 137;-   (A15) a protein comprising the amino acid sequence shown in SEQ ID    NO: 138;-   (A16) a protein comprising the amino acid sequence shown in SEQ ID    NO: 215;-   (A17) a protein comprising the amino acid sequence shown in SEQ ID    NO: 216;-   (A18) a protein comprising the amino acid sequence shown in SEQ ID    NO; 217;-   (A19) a protein comprising the amino acid sequence shown in SEQ ID    NO: 218;-   (A20) a protein comprising the amino acid sequence shown in SEQ ID    NO: 219;-   (A21) a protein comprising the amino acid sequence shown in SEQ ID    NO: 220;-   (A22) a protein comprising the amino acid sequence shown in SEQ ID    NO: 221;    -   (A23) a protein comprising the amino acid sequence shown in SEQ        ID NO: 222;-   (A24) a protein comprising the amino acid sequence shown in SEQ ID    NO: 223;-   (A25) a protein comprising the amino acid sequence shown in SEQ ID    NO: 224;-   (A26) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO:    136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219,    SEQ ID NO: 220, SEQ ID NO: 221 or SEQ ID NO: 223 or an amino acid    sequence having at least 90% sequence identity with an amino acid    sequence shown in any one of SEQ ID NO: 160, SEQ ID NO: 215, SEQ ID    NO: 216, SEQ ID NO: 218, SEQ ID NO: 222 or SEQ ID NO: 224;-   (A27) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    90% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 160,    SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 215, SEQ    ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO: 219, SEQ ID    NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 or SEQ ID    NO: 224; and-   (A28) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a DNA amplifiable by a polymerase    chain reaction with a primer comprising the nucleotide sequence    shown in any one of SEQ ID NOs: 124 to 128, a primer comprising the    nucleotide sequence shown in SEQ ID NO: 129 and as a template a    chromosomal DNA of Streptomyces phaeochromogenes, Streptomyces    testaceus, Streptomyces achromogenes, Streptomyces griseofuscus,    Streptomyces thermocoerulescens, Streptomyces nogalater,    Streptomyces tsusimaensis, Streptomyces glomerochromogenes,    Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces    griseus, Streptomyces lanatus, Streptomyces misawanensis,    Streptomyces pallidus, Streptomyces roseorubens, Streptomyces    rutgersensis, Streptomyces steffisburgensis or Saccharopolyspora    taberi;-   26. An analysis or detection kit comprising the antibody according    to the above 24;-   27. A DNA encoding a ferredoxin selected from the group consisting    of:-   (B1) a protein comprising an amino acid sequence shown in SEQ D NO:    12;-   (B2) a protein comprising an amino acid sequence shown in SEQ ID NO:    13;-   (B3) a protein comprising an amino acid sequence shown in SEQ ID NO:    14;-   (B4) a protein comprising an amino acid sequence shown in SEQ ID NO:    111;-   (B5) a ferredoxin comprising an amino acid sequence having at least    80% sequence identity with an amino acid sequence shown in any one    of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO 14 or SEQ ID NO 111;-   (B6) a ferredoxin comprising an amino acid sequence encoded by a    nucleotide sequence having at least 90% sequence identity with a    nucleotide sequence encoding an amino acid sequence shown in any one    of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO 14 or SEQ ID NO; 111;-   (B7) a protein comprising an amino acid sequence shown in SEQ ID NO:    149;-   (B8) a protein comprising an amino acid sequence shown in SEQ ID NO:    150;-   (B9) a protein comprising an amino acid sequence shown in SEQ ID NO:    151;-   (B10) a protein comprising an amino acid sequence shown in SEQ ID    NO: 152;-   (B11) a protein comprising an amino acid sequence shown in SEQ ID    NO: 153;-   (B12) a ferredoxin comprising an amino acid sequence having at least    80% sequence identity with an amino acid sequence shown in any one    of SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153,    SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ    ID NO: 250, SEQ ID NO: 251, or SEQ ID NO: 253 or an amino acid    sequence having at least 90% sequence identity with an amino acid    sequence shown in any one of SEQ ID NO: 150, SEQ ID NO: 252 or SEQ    ID NO: 254;-   (B13) a ferredoxin comprising an amino acid sequence encoded by a    nucleotide sequence having at least 90% sequence identity with a    nucleotide sequence encoding an amino acid sequence shown in any one    of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152,    SEQ ID NO: 153, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 248, SEQ    ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID    NO: 253 or SEQ ID NO: 254;-   (B14) a protein comprising the amino acid sequence shown in SEQ ID    NO: 245;-   (B15) a protein comprising the amino acid sequence shown in SEQ ID    NO: 247;-   (B16) a protein comprising the amino acid sequence shown in SEQ ID    NO: 248;-   (B17) a protein comprising the amino acid sequence shown in SEQ ID    NO: 249;-   (B18) a protein comprising the amino acid sequence shown in SEQ ID    NO: 250;-   (B19) a protein comprising the amino acid sequence shown in SEQ ID    NO: 251;-   (B20) a protein comprising the amino acid sequence shown in SEQ ID    NO: 252;-   (B21) a protein comprising the amino acid sequence shown in SEQ ID    NO: 253; and-   (B22) a protein comprising the amino acid sequence shown in SEQ ID    NO: 254;-   28. A DNA comprising a nucleotide sequence selected from the group    consisting of:-   (b1) a nucleotide sequence shown in SEQ ID NO: 15;-   (b2) a nucleotide sequence shown in SEQ ID NO: 16;-   (b3) a nucleotide sequence shown in SEQ ID NO: 17;-   (b4) a nucleotide sequence shown in SEQ ID NO: 112;-   (b5) a nucleotide sequence shown in SEQ ID NO: 154;-   (b6) a nucleotide sequence shown in SEQ ID NO; 155;-   (b7) a nucleotide sequence shown in SEQ ID NO: 156;-   (b8) a nucleotide sequence shown in SEQ ID NO: 157;-   (b9) a nucleotide sequence shown in SEQ ID NO: 158;-   (b10) a nucleotide sequence shown in SEQ ID NO: 255;-   (b11) a nucleotide sequence shown in SEQ ID NO: 257;-   (b12) a nucleotide sequence shown in SEQ ID NO: 258;-   (b13) a nucleotide sequence shown in SEQ ID NO: 259;-   (b14) a nucleotide sequence shown in SEQ ID NO: 260;-   (b15) a nucleotide sequence shown in SEQ ID NO: 261;-   (b16) a nucleotide sequence shown in SEQ ID NO: 262;-   (b17) a nucleotide sequence shown in SEQ ID NO: 263;-   (b18) a nucleotide sequence shown in SEQ ID NO: 264; and-   (b19) a nucleotide sequence having at least 90% sequence identity    with a nucleotide sequence shown in any one of SEQ ID NO: 15, SEQ ID    NO: 16, SEQ ID NO: 17, SEQ ID NO: 112, SEQ ID NO: 154, SEQ ID NO:    155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 255,    SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ    ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263 or SEQ ID NO: 264;-   29. A vector comprising a DNA according to the above 28;-   30. A transformant in which the DNA according to the above 28 is    introduced into a host cell;-   31. A ferredoxin selected from the group consisting of:-   (B1) a protein comprising an amino acid sequence shown in SEQ ID NO:    12;-   (B2) a protein comprising an amino acid sequence shown in SEQ ID NO:    13;-   (B3) a protein comprising an amino acid sequence shown in SEQ ID NO:    14;-   (B4) a protein comprising an amino acid sequence shown in SEQ ID NO:    111;-   (B5) a ferredoxin comprising an amino acid sequence having at least    80% sequence identity with an amino acid sequence shown in any one    of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO 14 or SEQ ID NO: 111;-   (B6) a ferredoxin comprising an amino acid sequence encoded by a    nucleotide sequence having at least 90% sequence identity with a    nucleotide sequence encoding an amino acid sequence shown in any one    of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO 14 or SEQ ID NO; 111;-   (B7) a protein comprising an amino acid sequence shown in SEQ ID NO:    149;-   (B8) a protein comprising an amino acid sequence shown in SEQ ID NO:    150;-   (B9) a protein comprising an amino acid sequence shown in SEQ ID NO;    151;-   (B10) a protein comprising an amino acid sequence shown in SEQ ID    NO; 152;-   (B11) a protein comprising an amino acid sequence shown in SEQ ID    NO: 153;-   (B12) a ferredoxin comprising an amino acid sequence having at least    80% sequence identity with an amino acid sequence shown in any one    of SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153,    SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NOT 249, SEQ    ID NO: 250, SEQ ID NO: 251, or SEQ ID NO: 253 or an amino acid    sequence having at least 90% sequence identity with an amino acid    sequence shown in any one of SEQ ID NO: 150, SEQ ID NO: 252 or SEQ    ID NO: 254;-   (B13) a ferredoxin comprising an amino acid sequence encoded by a    nucleotide sequence having at least 90% sequence identity with a    nucleotide sequence encoding an amino acid sequence shown in any one    of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152,    SEQ ID NO: 153, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 248, SEQ    ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID    NO: 253 or SEQ ID NO: 254;-   (B14) a protein comprising the amino acid sequence shown in SEQ ID    NO: 245;-   (B15) a protein comprising the amino acid sequence shown in SEQ ID    NO: 247;-   (B16) a protein comprising the amino acid sequence shown in SEQ ID    NO: 248;-   (B17) a protein comprising the amino acid sequence shown in SEQ ID    NO: 249;-   (B18) a protein comprising the amino acid sequence shown in SEQ ID    NO: 250;-   (B19) a protein comprising the amino acid sequence shown in SEQ ID    NO; 251;-   (B20) a protein comprising the amino acid sequence shown in SEQ ID    NO: 252;-   (B21) a protein comprising the amino acid sequence shown in SEQ ID    NO, 253; and-   (B22) a protein comprising the amino acid sequence shown in SEQ ID    NO: 254;-   32. A DNA comprising a nucleotide sequence selected from the group    consisting of:-   (ab1) a nucleotide sequence shown in SEQ ID NO: 9;-   (ab2) a nucleotide sequence shown in SEQ ID NO: 10;-   (ab3) a nucleotide sequence shown in SEQ ID NO: 11;-   (ab4) a nucleotide sequence shown in SEQ ID NO: 110;-   (ab5) a nucleotide sequence shown in SEQ ID NO: 144;-   (ab6) a nucleotide sequence shown in SEQ ID NO: 145;-   (ab7) a nucleotide sequence shown in SEQ ID NO: 146;-   (ab8) a nucleotide sequence shown in SEQ ID NO: 147;-   (ab9) a nucleotide sequence shown in SEQ ID NO; 148;-   (ab10) a nucleotide sequence shown in SEQ ID NO: 235;-   (ab11) a nucleotide sequence shown in SEQ ID NO: 236;-   (ab12) a nucleotide sequence shown in SEQ ID NO: 237;-   (ab13) a nucleotide sequence shown in SEQ ID NO: 238;-   (ab14) a nucleotide sequence shown in SEQ ID NO: 239;-   (ab15) a nucleotide sequence shown in SEQ ID NO: 240;-   (ab16) a nucleotide sequence shown in SEQ ID NO; 241;-   (ab17) a nucleotide sequence shown in SEQ ID NO: 242;-   (ab18) a nucleotide sequence shown in SEQ ID NO: 243; and-   (ab19) a nucleotide sequence shown in SEQ ID NO: 244;-   33. A vector comprising the DNA according to the above 32;-   34. A transformant in which the DNA according to the above 32 is    introduced into a host cell;-   35. The transformant according to the above 34, wherein the host    cell is a microorganism cell or a plant cell;-   36. A method of producing a transformant comprising a step of    introducing into a host cell the DNA according to the above 32;-   37. A method of producing a protein having the ability to convert a    compound of formula (II) to a compound of formula (III), said method    comprising a step of culturing the transformant according to the    above 34 and recovering the produced said protein;-   38. A method of controlling weeds comprising a step of applying a    compound to a cultivation area of a plant expressing at least one    herbicide metabolizing protein selected from the group consisting    of:-   (A1) a protein comprising the amino acid sequence shown in SEQ ID    NO: 1;-   (A2) a protein comprising the amino acid sequence shown in SEQ ID    NO: 2;-   (A3) a protein comprising the amino acid sequence shown in SEQ ID    NO: 3;-   (A4) a protein comprising the amino acid sequence shown in SEQ ID    NO: 108;-   (A5) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2,    SEQ ID NO: 3 or SEQ ID NO 108;-   (A6) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    80% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID    NO: 3 or SEQ ID NO: 108;-   (A7) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a DNA that hybridizes, under    stringent conditions, to a DNA comprising a nucleotide sequence    encoding an amino acid sequence shown in any one of SEQ ID NO: 1,    SEQ ID NO: 2, SEQ ID NO; 3 or SEQ ID NO: 108;-   (A8) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a DNA amplifiable by a polymerase    chain reaction with a primer comprising a nucleotide sequence shown    in SEQ ID NO; 129, a primer comprising a nucleotide sequence shown    in any one of SEQ ID NOs: 124 to 128, and as a template a chromosome    of a microorganism belonging to Streptomyces or Saccharopolyspora;-   (A9) a protein comprising an amino acid sequence shown in SEQ ID NO:    4;-   (A11) a protein comprising the amino acid sequence shown in SEQ ID    NO: 159;-   (A12) a protein comprising the amino acid sequence shown in SEQ ID    NO: 160;-   (A13) a protein comprising the amino acid sequence shown in SEQ ID    NO; 136;-   (A14) a protein comprising the amino acid sequence shown in SEQ ID    NO: 137;-   (A15) a protein comprising the amino acid sequence shown in SEQ ID    NO: 138;-   (A16) a protein comprising the amino acid sequence shown in SEQ ID    NO: 215;-   (A17) a protein comprising the amino acid sequence shown in SEQ ID    NO: 216;-   (A18) a protein comprising the amino acid sequence shown in SEQ ID    NO: 217;-   (A19) a protein comprising the amino acid sequence shown in SEQ ID    NO: 218;-   (A20) a protein comprising the amino acid sequence shown in SEQ ID    NO: 219;-   (A21) a protein comprising the amino acid sequence shown in SEQ ID    NO: 220;-   (A22) a protein comprising the amino acid sequence shown in SEQ ID    NO; 221;-   (A23) a protein comprising the amino acid sequence shown in SEQ ID    NO: 222;-   (A24) a protein comprising the amino acid sequence shown in SEQ ID    NO: 223;-   (A25) a protein comprising the amino acid sequence shown in SEQ ID    NO: 224;-   (A26) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO:    136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219,    SEQ ID NO: 220, SEQ ID NO: 221 or SEQ ID NO: 223 or an amino acid    sequence having at least 90% sequence identity with an amino acid    sequence shown in any one of SEQ ID NO: 160, SEQ ID NO: 215, SEQ ID    NO: 216, SEQ ID NO: 218, SEQ ID NO: 222 or SEQ ID NO: 224; and-   (A27) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    90% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 160,    SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 215, SEQ    ID NO; 216, SEQ ID NO; 217, SEQ ID NO:218, SEQ ID NO, 219, SEQ ID    NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 or SEQ ID    NO: 224,    wherein said compound is a compound of formula (I):    wherein in formula (I) G represents a group shown in any one of the    following G-1 to G-9:    where in G-1 to G-9,    -   X represents an oxygen atom or sulfur atom;    -   Y represents an oxygen atom or sulfur atom;    -   R¹ represents a hydrogen atom or halogen atom;    -   R² represents a hydrogen atom, C₁-C₈ alkyl group, C₁-C₈        haloalkyl group, halogen atom, hydroxyl group, —OR⁹ group, —SH        group, —S(O)pR⁹ group, —COR⁹ group, —CO₂R⁹ group, —C(O)SR⁹        group, —C(O)NR¹¹R¹² group, —CONH₂ group, —CHO group, —CR⁹═NOR¹⁸        group, —CH═CR¹⁹CO₂R⁹ group, —CH₂CHR¹⁹CO₂R⁹ group, —CO₂N═CR¹³R¹⁴        group, nitro group, cyano group, —NHSO₂R¹⁵ group, —NHSO₂NHR¹⁵        group, —NR⁹R²⁰ group, —NH₂ group or phenyl group that may be        substituted with one or more C₁-C₄ alkyl groups which may be the        same or different;    -   p represents 0, 1 or 2;    -   R³ represents C₁-C₂ alkyl group, C₁-C₂ haloalkyl group, —OCH₃        group, —SCH₃ group, —OCHF₂ group, halogen atom, cyano group,        nitro group or C₁-C₃ alkoxy group substituted with a phenyl        group which may be substituted on the ring with at least one        substituent selected from a halogen atom, C₁-C₃ alkyl group,        C₁-C₃ haloalkyl group, OR¹⁸ group, NR¹¹R²⁸ group, SR²⁸ group,        cyano group, CO₂R²⁸ group and nitro group;    -   R⁴ represents a hydrogen atom, C₁-C₃ alkyl group or C₁-C₃        haloalkyl group;    -   R⁵ represents a hydrogen atom, C₁-C₃ alkyl group, C₁-C₃        haloalkyl group, cyclopropyl group, vinyl group, C₂ alkynyl        group, cyano group, —C(O)R²⁰ group, —CO₂R²⁰ group, —C(O)NR²⁰R²¹        group, —CHR¹⁶R¹⁷CN group, —CR¹⁶R¹⁷C(O)R²⁰ group, —C¹⁶R¹⁷CO₂R²⁰        group, —CR¹⁶R¹⁷C(O)NR²⁰R²¹ group, CHR¹⁶OH group, —CHR¹⁶OC(O)R²⁰        group or —OCHR¹⁶OC(O)NR²⁰R²¹ group, or, when G represents G-2 or        G-6, R⁴ and R⁵ may represent C═O group together with the carbon        atom to which they are attached;    -   R⁶ represents C₁-C₆ alkyl group, C₁-C₆ haloalkyl group, C₂-C₆        alkoxyalkyl group, C₃-C₆alkenyl group or C₃-C₆ alkynyl group;    -   R⁷ represents a hydrogen atom, C₁-C₆ alkyl group, C₁-C₆        haloalkyl group, halogen atom, —S(O)₂(C₁-C₆ alkyl) group or        —C(═O)R²² group;    -   R⁸ represents a hydrogen atom, C₁-C₈ alkyl group, C₃-C₈        cycloalkyl group, C₃-C₈ alkenyl group, C₃-C₈ alkynyl group,        C₁-C₈ haloalkyl group, C₂-C₈ alkoxyalkyl group, C₃-C₈        alkoxyalkoxyalkyl group, C₃-C₈ haloalkynyl group, C₃-C₈        haloalkenyl group, C₁-C₈ alkylsulfonyl group, C₁-C₈        haloalkylsulfonyl group, C₃-C₈ alkoxycarbonylalkyl group,        —S(O)₂NH(C₁-C₈ alkyl) group, —C(O)R²³ group or benzyl group        which may be substituted with R²⁴ on the phenyl ring;    -   R⁹ represents C₁-C₈ alkyl group, C₃-C₈ cycloalkyl group, C₃-C₈        alkenyl group, C₃-C₈ alkynyl group, C₁-C₈ haloalkyl group, C₂-C₈        alkoxyalkyl group, C₂-C₈ alkylthioalkyl group, C₂-C₈        alkylsulfinylalkyl group, C₂-C₈ alkylsulfonylalkyl group, C₄-C₈        alkoxyalkoxyalkyl group, C₄-C₈ cycloalkylalkyl group, C₄-C₈        cycloalkoxyalkyl group, C₄-C₈ alkenyloxyalkyl group, C₄-C₈        alkynyloxyalkyl group, C₃-C₈ haloalkoxyalkyl group, C₄-C₈        haloalkenyloxyalkyl group, C₄-C₈ haloalkynyloxyalkyl group,        C₄-C₈ cycloalkylthioalkyl group, C₄-C₈ alkenylthioalkyl group,        C₄-C₈ alkynylthioalkyl group, C₁-C₄ alkyl group substituted with        a phenoxy group which may be substituted on the ring with at        least one substituent selected from a halogen atom, C₁-C₃ alkyl        group and C₁-C₃ haloalkyl group, C₁-C₄ alkyl group substituted        with a benzyloxy group which may be substituted on the ring with        at least one substituent selected from a halogen atom, C₁-C₃        alkyl group and C₁-C₃ haloalkyl group, C₄-C₈ trialkylsyrylalkyl        group, C₂-C₈ cyanoalkyl group, C₃-C₈ halocycloalkyl group, C₃-C₈        haloalkenyl group, C₅-C₈ alkoxyalkenyl group, C₅-C₈        haloalkoxyalkenyl group, C₅-C₈ alkylthioalkenyl group, C₃-C₈        haloalkynyl group, C₅-C₈ alkoxyalkynyl group, C₅-C₈        haloalkoxyalkynyl group, C₅-C₈ alkylthioalkynyl group, C₂-C₈        alkylcarbonyl group, benzyl group which may be substituted on        the ring with at least one substituent selected from a halogen        atom, C₁-C₃ alkyl group, C₁-C₃ haloalkyl group, —OR²⁸ group,        —NR¹¹R²⁸ group, —SR²⁸ group, cyano group, —CO₂R²⁸ group and        nitro group, —CR¹⁶R¹⁷COR¹⁰ group, —CR¹⁶R¹⁷CO₂R²⁰ group,        —CR¹⁶R¹⁷P(O)(OR¹⁰)₂ group, —CR¹⁶R¹⁷P(S)(OR¹⁰)₂ group,        —CR¹⁶R¹⁷C(O)NR¹¹R¹² group, —CR¹⁶R¹⁷C(O)NH₂ group,        —C(═CR²⁶R²⁷)COR¹⁰ group, —C(═CR²⁶R²⁷)CO₂R²⁰ group,        —(═CR²⁶R²⁷)P(O)(OR¹⁰)₂ group, —C(═CR²⁶R²⁷)P(S)(OR¹⁰)₂ group,        —C(═CR²⁶R²⁷)C(O)NR¹¹R¹² group, —C(═CR²⁶R²⁷)C(O)NH₂ group, or any        one of rings shown in Q-1 to Q-7:        which may be substituted on the ring with at least one        substituent selected from a halogen atom, C₁-C₆ alkyl group,        C₁-C₆ haloalkyl group, C₂-C₆ alkenyl group, C₂-C₆ haloalkenyl        group, C₂-C₆ alkynyl group, C₃-C₆ haloalkynyl group, C₂-C₈        alkoxyalkyl group, —OR²⁸ group, —SR²⁸ group, NR¹¹R²⁸ group,        C₃-C₈ alkoxycarbonylalkyl group, C₂-C₄ carboxyalkyl group,        —CO₂R²⁸ group and cyano group;    -   R¹⁰ represents a C₁-C₆ alkyl group, C₂-C₆ alkenyl group, C₃-C₆        alkynyl group or tetrahydrofuranyl group;    -   R¹¹ and R¹³ independently represent a hydrogen atom or C₁-C₄        alkyl group;    -   R¹² represents C₁-C₆ alkyl group, C₃-C₆ cycloalkyl group, C₃-C₆        alkenyl group, C₃-C₆ akynyl group, C₂-C₆ alkoxyalkyl group,        C₁-C₆ haloalkyl group, C₃-C₆ haloalkenyl group, C₃-C₆        haloalkynyl group, phenyl group which may be substituted on the        ring with at least one substituent selected from a halogen atom,        C₁-C₄ alkyl group and C₁-C₄ alkoxy group or —CR¹⁶R¹⁷CO₂R²⁵        group; or,    -   R¹¹ and R¹² together may represent —(CH₂)₅—, —(CH₂)₄— or        —CH₂CH₂OCH₂CH₂—, or in that case the resulting ring may be        substituted with a substituent selected from a C₁-C₃ alkyl        group, a phenyl group and benzyl group;    -   R¹⁴ represents a C₁-C₄ alkyl group or phenyl group which may be        substituted on the ring with a substituent selected from a        halogen atom, C₁-C₃ alkyl group and C₁-C₃ haloalkyl group; or,    -   R¹³ and R¹⁴ may represent C₃-C₈ cycloalkyl group together with        the carbon atom to which they are attached;    -   R¹⁵ represents C₁-C₄ alkyl group, C₁-C₄ haloalkyl group or C₃-C₆        alkenyl group;    -   R¹⁶ and R¹⁷ independently represent a hydrogen atom or C₁-C₄        alkyl group, C₁-C₄ haloalkyl group, C₂-C₄ alkenyl group, C₂-C₄        haloalkenyl group, C₂-C₄ alkynyl group, C₃-C₄ haloalkynyl group;        or,    -   R¹⁶ and R¹⁷ may represent C₃-C₆ cycloalkyl group with the carbon        atom to which they are attached, or the ring thus formed may be        substituted with at least one substituent selected from a        halogen atom, a C₁-C₃ alkyl group and C₁-C₃ haloalkyl group;    -   R¹⁸ represents a hydrogen atom, C₁-C₆ alkyl group, C₃-C₆ alkenyl        group or C₃-C₆ alkynyl group;    -   R¹⁹ represents a hydrogen atom, C₁-C₄ alkyl group or halogen        atom,    -   R²⁰ represents a hydrogen atom, C₁-C₆ alkyl group, C₃-C₆        cycloalkyl group, C₃-C₆ alkenyl group, C₃-C₆ alkynyl group,        C₂-C₆ alkoxyalkyl group, C₁-C₆ haloalkyl group, C₃-C₆        haloalkenyl group, C₃-C₆ haloalkynyl group, phenyl group which        may be substituted on the ring with at least one substituent        selected from a halogen atom, C₁-C₄ alkyl group and —OR²⁸ group,        or —CR¹⁶R¹⁷CO₂R²⁵ group;    -   R²¹ represents a hydrogen atom, C₁-C₂ alkyl group or —CO₂(C₁-C₄        alkyl) group;    -   R²² represents a hydrogen atom, C₁-C₆ alkyl group, C₁-C₆ alkoxy        group or NH(C₁-C₆ alkyl) group;    -   R²³ represents C₁-C₆ alkyl group, C₁-C₆ haloalkyl group, C₁-C₆        alkoxy group, NH(C₁-C₆ alkyl) group, benzyl group, C₂-C₈        dialkylamino group or phenyl group which may be substituted with        R²⁴;    -   R²⁴ represents C₁-C₆ alkyl group, 1 to 2 halogen atoms, C₁-C₆        alkoxy group or CF₃ group;    -   R²⁵ represents C₁-C₆ alkyl group, C₁-C₆ haloalkyl group, C₃-C₆        alkenyl group, C₃-C₆ haloalkenyl group, C₃-C₆ alkynyl group or        C₃-C₆ haloalkynyl group;    -   R²⁶ and R²⁷ each represent independently a hydrogen atom, C₁-C₄        alkyl group, C₁-C₄ haloalkyl group, C₂-C₄ alkenyl group, C₂-C₄        haloalkenyl group, C₂-C₄ alkynyl group, C₃-C₄ haloalkynyl group,        —OR²⁸ group, —NHR²⁸ group, or —SR²⁸ group; or,    -   R²⁶ and R²⁷ may represent C₃-C₈ cycloalkyl group with the carbon        atom to which they are attached, or each of the ring thus formed        may be substituted with at least one substituent selected from a        halogen atom, C₁-C₃ alkyl group and C₁-C₃ haloalkyl group; and,    -   R²⁸ represents a hydrogen atom, C₁-C₆ alkyl group, C₁-C₄        haloalkyl group, C₃-C₆ alkenyl group, C₃-C₆ haloalkenyl group,        C₃-C₆ alkynyl group, C₃-C₆ haloalkynyl group, C₂-C₄ carboxyalkyl        group, C₃-C₈ alkoxycarbonylalkyl group, C₃-C₈        haloalkoxycarbonylalkyl group, C₅-C₉ alkenyloxycabonylalkyl        group, C₅-C₉ haloalkenyloxycabonylalkyl group, C₅-C₉        alkynyloxycabonylalkyl group, C₅-C₉ haloalkynyloxycabonylalkyl        group, C₅-C₉ cycloalkoxycabonylalkyl group or C₅-C₉        halocycloalkoxycabonylalkyl group;-   39. A method of controlling weeds comprising a step of applying a    compound to a cultivation area of a plant expressing at least one    protein selected from the group consisting of:-   (A1) a protein comprising the amino acid sequence shown in SEQ ID    NO: 1;-   (A2) a protein comprising the amino acid sequence shown in SEQ ID    NO: 2;-   (A3) a protein comprising the amino acid sequence shown in SEQ ID    NO: 3;-   (A4) a protein comprising the amino acid sequence shown in SEQ ID    NO: 108;-   (A5) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III) and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2,    SEQ ID NO: 3 or SEQ ID NO: 108;-   (A6) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    80% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID    NO: 3 or SEQ ID NO: 108;-   (A11) a protein comprising the amino acid sequence shown in SEQ ID    NO: 159;-   (A12) a protein comprising the amino acid sequence shown in SEQ ID    NO: 160;-   (A13) a protein comprising the amino acid sequence shown in SEQ ID    NO, 136;-   (A14) a protein comprising the amino acid sequence shown in SEQ ID    NO: 137;-   (A15) a protein comprising the amino acid sequence shown in SEQ ID    NO: 138;-   (A16) a protein comprising the amino acid sequence shown in SEQ ID    NO: 215;-   (A17) a protein comprising the amino acid sequence shown in SEQ ID    NO: 216;-   (A18) a protein comprising the amino acid sequence shown in SEQ ID    NO: 217;-   (A19) a protein comprising the amino acid sequence shown in SEQ ID    NO: 218;-   (A20) a protein comprising the amino acid sequence shown in SEQ ID    NO: 219;-   (A21) a protein comprising the amino acid sequence shown in SEQ ID    NO: 220;-   (A22) a protein comprising the amino acid sequence shown in SEQ ID    NO: 221;-   (A23) a protein comprising the amino acid sequence shown in SEQ ID    NO: 222;-   (A24) a protein comprising the amino acid sequence shown in SEQ ID    NO: 223;-   (A25) a protein comprising the amino acid sequence shown in SEQ ID    NO; 224;-   (A26) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO:    136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219,    SEQ ID NO: 220, SEQ ID NO: 221 or SEQ ID NO: 223 or an amino acid    sequence having at least 90% sequence identity with an amino acid    sequence shown in any one of SEQ ID NO: 160, SEQ ID NO: 215, SEQ ID    NO; 216, SEQ ID NO: 218, SEQ ID NO: 222 or SEQ ID NO: 224;-   (A27) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    90% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 160,    SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 215, SEQ    ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO: 219, SEQ ID    NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 or SEQ ID    NO: 224; and-   (A28) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a DNA amplifiable by a polymerase    chain reaction with a primer comprising the nucleotide sequence    shown in any one of SEQ ID NOs: 124 to 128, a primer comprising the    nucleotide sequence shown in SEQ ID NO: 129 and as a template a    chromosomal DNA of Streptomyces phaeochromogenes, Streptomyces    testaceus, Streptomyces achromogenes, Streptomyces griseofuscus,    Streptomyces thermocoerulescens, Streptomyces nogalater,    Streptomyces tsusimaensis, Streptomyces glomerochromogenes,    Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces    griseus, Streptomyces lanatus, Streptomyces misawanensis,    Streptomyces pallidus, Streptomyces roseorubens, Streptomyces    rutgersensis, Streptomyces steffisburgensis or Saccharopolyspora    taberi;-   40. A method of evaluating the resistance of a cell to a compound of    formula (I), said method comprising:    -   (1) a step of contacting said compound with a cell expressing at        least one herbicide metabolizing protein selected from the group        consisting of-   (A1) a protein comprising the amino acid sequence shown in SEQ ID    NO: 1;-   (A2) a protein comprising the amino acid sequence shown in SEQ ID    NO: 2;-   (A3) a protein comprising the amino acid sequence shown in SEQ ID    NO: 3;-   (A4) a protein comprising the amino acid sequence shown in SEQ ID    NO: 108;-   (A5) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III) and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2,    SEQ ID NO: 3 or SEQ ID NO: 108;-   (A6) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    80% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID    NO: 3 or SEQ ID NO: 108;-   (A7) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a DNA that hybridizes, under    stringent conditions, to a DNA comprising a nucleotide sequence    encoding an amino acid sequence shown in any one of SEQ ID NO: 1,    SEQ ID NO: 2, SEQ ID NO; 3 or SEQ ID NO: 108;-   (A8) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a DNA amplifiable by a polymerase    chain reaction with a primer comprising a nucleotide sequence shown    in SEQ ID NO; 129, a primer comprising a nucleotide sequence shown    in any one of SEQ ID NOs: 124 to 128, and as a template a chromosome    of a microorganism belonging to Streptomyces or Saccharopolyspora;-   (A9) a protein comprising an amino acid sequence shown in SEQ ID NO:    4;-   (A11) a protein comprising the amino acid sequence shown in SEQ ID    NO: 159;-   (A12) a protein comprising the amino acid sequence shown in SEQ ID    NO: 160;-   (A13) a protein comprising the amino acid sequence shown in SEQ ID    NO: 136;-   (A14) a protein comprising the amino acid sequence shown in SEQ ID    NO: 137;-   (A15) a protein comprising the amino acid sequence shown in SEQ ID    NO: 138;-   (A16) a protein comprising the amino acid sequence shown in SEQ ID    NO: 215;-   (A17) a protein comprising the amino acid sequence shown in SEQ ID    NO: 216;-   (A18) a protein comprising the amino acid sequence shown in SEQ ID    NO: 217;-   (A19) a protein comprising the amino acid sequence shown in SEQ ID    NO: 218;-   (A20) a protein comprising the amino acid sequence shown in SEQ ID    NO: 219;-   (A21) a protein comprising the amino acid sequence shown in SEQ ID    NO: 220;-   (A22) a protein comprising the amino acid sequence shown in SEQ ID    NO: 221;-   (A23) a protein comprising the amino acid sequence shown in SEQ ID    NO: 222;-   (A24) a protein comprising the amino acid sequence shown in SEQ ID    NO: 223;-   (A25) a protein comprising the amino acid sequence shown in SEQ ID    NO: 224;-   (A26) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO:    136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219,    SEQ ID NO: 220, SEQ ID NO: 221 or SEQ ID NO: 223 or an amino acid    sequence having at least 90% sequence identity with an amino acid    sequence shown in any one of SEQ ID NO: 160, SEQ ID NO: 215, SEQ ID    NO: 216, SEQ ID NO: 218, SEQ ID NO: 222 or SEQ ID NO: 224; and-   (A27) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    90% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 160,    SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 13, SEQ ID NO: 215, SEQ    ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO: 219, SEQ ID    NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 or SEQ ID    NO: 224; and    -   (2) a step of evaluating the degree of damage to the cell which        contacted the compound in the above step (1);-   41. The method according to the above 40, wherein the cell is a    microorganism cell or plaint cell;-   42. A method of selecting a cell resistant to a compound of formula    (1), said method comprising a step of selecting a cell based on the    resistance evaluated in the method according to the above 40;-   43. The cell resistant to herbicide selected by the method according    to the above 42, or the culture thereof;-   44. A method of evaluating the resistance of a plant to a compound    of formula (1), said method comprising:    -   (1) a step of contacting said compound with a plant expressing        at least one herbicide metabolizing protein selected from the        group consisting of:-   (A1) a protein comprising the amino acid sequence shown in SEQ ID    NO: 1;-   (A2) a protein comprising the amino acid sequence shown in SEQ ID    NO: 2;-   (A3) a protein comprising the amino acid sequence shown in SEQ ID    NO: 3;-   (A4) a protein comprising the amino acid sequence shown in SEQ ID    NO: 108;-   (A5) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III) and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequences shown in any one of SEQ ID NO: 1, SEQ ID NO: 2,    SEQ ID NO: 3 or SEQ ID NO: 108;-   (A6) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    80% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID    NO: 3 or SEQ ID NO: 108;-   (A7) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a DNA that hybridizes, under    stringent conditions, to a DNA comprising a nucleotide sequence    encoding an amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2,    SEQ ID NO: 3 or SEQ ID NO: 108;-   (A8) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor a compound of    formula (I) to a compound of formula (III), and comprising an amino    acid sequence encoded by a DNA amplifiable by a polymerase chain    reaction with a primer comprising a nucleotide sequence shown in SEQ    ID NO: 129, a primer comprising a nucleotide sequence shown in any    one of SEQ ID NOs; 124 to 128, and as a template a chromosome of a    microorganism belonging to Streptomyces or Saccharopolyspora;-   (A9) a protein comprising an amino acid sequence shown in SEQ ID NO:    4;-   (A11) a protein comprising the amino acid sequence shown in SEQ ID    NO: 159;-   (A12) a protein comprising the amino acid sequence shown in SEQ ID    NO: 160;-   (A13) a protein comprising the amino acid sequence shown in SEQ ID    NO: 136;-   (A14) a protein comprising the amino acid sequence shown in SEQ ID    NO: 137;-   (A15) a protein comprising the amino acid sequence shown in SEQ ID    NO: 138;-   (A16) a protein comprising the amino acid sequence shown in SEQ ID    NO: 215;-   (A17) a protein comprising the amino acid sequence shown in SEQ ID    NO: 216;-   (A18) a protein comprising the amino acid sequence shown in SEQ ID    NO: 217;-   (A19) a protein comprising the amino acid sequence shown in SEQ ID    NO: 218;-   (A20) a protein comprising the amino acid sequence shown in SEQ ID    NO: 219;-   (A21) a protein comprising the amino acid sequence shown in SEQ ID    NO: 220;-   (A22) a protein comprising the amino acid sequence shown in SEQ ID    NO: 221;-   (A23) a protein comprising the amino acid sequence shown in SEQ ID    NO: 222;-   (A24) a protein comprising the amino acid sequence shown in SEQ ID    NO: 223;-   (A25) a protein comprising the amino acid sequence shown in SEQ ID    NO: 224;-   (A26) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO:    136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219,    SEQ ID NO: 220, SEQ ID NO: 221 or SEQ ID NO: 223 or an amino acid    sequence having at least 90% sequence identity with an amino acid    sequence shown in any one of SEQ ID NO: 160, SEQ ID NO: 215, SEQ ID    NO: 216, SEQ ID NO: 218, SEQ ID NO: 222 or SEQ ID NO: 224; and-   (A27) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor a compound of    formula (II) to a compound of formula (III), and comprising an amino    acid sequence encoded by a nucleotide sequence having at least 90%    sequence identity with a nucleotide sequence encoding an amino acid    sequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID    NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 215, SEQ ID NO:    216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO: 219, SEQ ID NO: 220,    SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 or SEQ ID NO: 224;    and    -   (2) a step of evaluating the degree of damage to the plant which        contacted the compound described in step (1);-   45. A method of selecting a plant resistant to a compound of formula    C), said method comprising a step of selecting a plant based on the    resistance evaluated in the method according to the above 44;-   46. A herbicidally resistant plant selected from the method    according to the above 45, or the progeny thereof;-   47. A method of treating a compound of formula (1), said method    comprising reacting said compound in the presence of an electron    transport system containing an electron donor, with at least one    herbicide metabolizing protein selected from the group consisting    of:-   (A1) a protein comprising the amino acid sequence shown in SEQ ID    NO: 1;-   (A2) a protein comprising the amino acid sequence shown in SEQ ID    NO: 2;-   (A3) a protein comprising the amino acid sequence shown in SEQ ID    NO: 3;-   (A4) a protein comprising the amino acid sequence shown in SEQ ID    NO: 108;-   (A5) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III) and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2,    SEQ ID NO; 3 or SEQ ID NO: 108;-   (A6) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    80% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID    NO: 3 or SEQ ID NO: 108;-   (A7) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a DNA that hybridizes, under    stringent conditions, to a DNA comprising a nucleotide sequence    encoding an amino acid sequence shown in any one of SEQ ID NO: 1,    SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 108;-   (A8) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a DNA amplifiable by a polymerase    chain reaction with a primer comprising a nucleotide sequence shown    in SEQ ID NO: 129, a primer comprising a nucleotide sequence shown    in any one of SEQ ID NOs: 124 to 128, and as a template a chromosome    of a microorganism belonging to Streptomyces or Saccharopolyspora;-   (A9) a protein comprising an amino acid sequence shown in SEQ ID NO:    4;-   (A11) a protein comprising the amino acid sequence shown in SEQ ID    NO: 159;-   (A12) a protein comprising the amino acid sequence shown in SEQ ID    NO: 160;-   (A13) a protein comprising the amino acid sequence shown in SEQ ID    NO: 136;-   (A14) a protein comprising the amino acid sequence shown in SEQ ID    NO: 137;-   (A15) a protein comprising the amino acid sequence shown in SEQ ID    NO: 138;-   (A16) a protein comprising the amino acid sequence shown in SEQ ID    NO: 215;-   (A17) a protein comprising the amino acid sequence shown in SEQ ID    NO: 216;-   (A18) a protein comprising the amino acid sequence shown in SEQ ID    NO: 217;-   (A19) a protein comprising the amino acid sequence shown in SEQ ID    NO: 218;-   (A20) a protein comprising the amino acid sequence shown in SEQ ID    NO: 219;-   (A21) a protein comprising the amino acid sequence shown in SEQ ID    NO: 220;-   (A22) a protein comprising the amino acid sequence shown in SEQ ID    NO: 221;-   (A23) a protein comprising the amino acid sequence shown in SEQ ID    NO 222;-   (A24) a protein comprising the amino acid sequence shown in SEQ ID    NO: 223;-   (A25) a protein comprising the amino acid sequence shown in SEQ ID    NO: 224;-   (A26) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor a compound of    formula (II) to a compound of formula (III), and comprising an amino    acid sequence having at least 80% sequence identity with an amino    acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 136,    SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219, SEQ    ID NO: 220, SEQ ID NO: 221 or SEQ ID NO: 223 or an amino acid    sequence having at least 90% sequence identity with an amino acid    sequence shown in any one of SEQ ID NO: 160, SEQ ID NO: 215, SEQ ID    NO: 216, SEQ ID NO: 218, SEQ ID NO: 222 or SEQ ID NO: 224; and-   (A27) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    90% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 160,    SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 215, SEQ    ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO: 219, SEQ ID    NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 or SEQ ID    NO: 224;-   48. the method according to the above 47, wherein reacting the    compound with the herbicide metabolizing protein by contacting the    compound with a transformant in which a DNA encoding the herbicide    metabolizing protein is introduced into a host cell in a position    enabling its expression in said cell;-   49. Use for treating the compound of formula (I) of a herbicide    metabolizing protein selected from the group consisting of:-   (A1) a protein comprising the amino acid sequence shown in SEQ ID    NO, 1;-   (A2) a protein comprising the amino acid sequence shown in SEQ ID    NO: 2;-   (A3) a protein comprising the amino acid sequence shown in SEQ ID    NO: 3;-   (A4) a protein comprising the amino acid sequence shown in SEQ ID    NO: 108;-   (A5) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III) and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2,    SEQ ID NO: 3 or SEQ ID NO: 108;-   (A6) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    80% sequence identity with a nucleotide sequence encoding any one of    the amino acid sequences shown in any one of SEQ ID NO: 1, SEQ ID    NO: 2, SEQ ID NO: 3 or SEQ ID NO: 108;-   (A7) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a DNA that hybridizes, under    stringent conditions, to a DNA comprising a nucleotide sequence    encoding an amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2,    SEQ ID NO: 3 or SEQ ID NO; 108;-   (A8) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a DNA amplifiable by a polymerase    chain reaction with a primer comprising a nucleotide sequence shown    in SEQ ID NO: 129, a primer comprising a nucleotide sequence shown    in any one of SEQ ID NOs: 124 to 128, and as a template chromosome    of a microorganism belonging to Streptomyces or Saccharopolyspora;-   (A9) a protein comprising an amino acid sequence shown in SEQ ID NO:    4;-   (A11) a protein comprising the amino acid sequence shown in SEQ ID    NO: 159;-   (A12) a protein comprising the amino acid sequence shown in SEQ ID    NO: 160;-   (A13) a protein comprising the amino acid sequence shown in SEQ ID    NO: 136;-   (A14) a protein comprising the amino acid sequence shown in SEQ ID    NO: 137;-   (A15) a protein comprising the amino acid sequence shown in SEQ ID    NO: 138;-   (A16) a protein comprising the amino acid sequence shown in SEQ ID    NO: 215;-   (A17) a protein comprising the amino acid sequence shown in SEQ ID    NO: 216;-   (A18) a protein comprising the amino acid sequence shown in SEQ ID    NO: 217;-   (A19) a protein comprising the amino acid sequence shown in SEQ ID    NO: 218;-   (A20) a protein comprising the amino acid sequence shown in SEQ ID    NO: 219;-   (A21) a protein comprising the amino acid sequence shown in SEQ ID    NO: 220;-   (A22) a protein comprising the amino acid sequence shown in SEQ ID    NO: 221;-   (A23) a protein comprising the amino acid sequence shown in SEQ ID    NO: 222;-   (A24) a protein comprising the amino acid sequence shown in SEQ ID    NO: 223;-   (A25) a protein comprising the amino acid sequence shown in SEQ ID    NO: 224;-   (A26) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO;    136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219,    SEQ ID NO: 220, SEQ ID NO: 221 or SEQ ID NO: 223 or an amino acid    sequence having at least 90% sequence identity with an amino acid    sequence shown in anyone of SEQ ID NO: 160, SEQ ID NO: 215, SEQ ID    NO: 216, SEQ ID NO: 218, SEQ ID NO: 222 or SEQ ID NO: 224; and-   (A27) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    90% sequence identity with a nucleotide sequence encoding the amino    acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 160,    SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 215, SEQ    ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO: 219, SEQ ID    NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 or SEQ ID    NO: 224; and-   50. Use for treating a compound of formula (1) of a polynucleotide    encoding a herbicide metabolizing protein selected from the group    consisting of-   (A1) a protein comprising the amino acid sequence shown in SEQ ID    NO: 1;-   (A2) a protein comprising the amino acid sequence shown in SEQ ID    NO: 2;-   (A3) a protein comprising the amino acid sequence shown in SEQ ID    NO: 3;-   (A4) a protein comprising the amino acid sequence shown in SEQ ID    NO, 108;-   (A5) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III) and comprising an    amino acid sequence having at least 80% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2,    SEQ ID NO: 3 or SEQ ID NO: 108;-   (A6) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor a compound of    formula (II) to a compound of formula (III), and comprising an amino    acid sequence encoded by a nucleotide sequence having at least 80%    sequence identity with a nucleotide sequence encoding an amino acid    sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:    3 or SEQ ID NO: 108;-   (A7) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a DNA that hybridizes, under    stringent conditions, to a DNA comprising a nucleotide sequence    encoding an amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2,    SEQ ID NO: 3 or SEQ ID NO: 108;-   (A8) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a DNA amplifiable by a polymerase    chain reaction with a primer comprising a nucleotide sequence shown    in SEQ ID NO: 129, a primer comprising a nucleotide sequence shown    in any one of SEQ ID NOs: 124 to 128, and as a template a chromosome    of a microorganism belonging to Streptomyces or Saccharopolyspora;-   (A9) a protein comprising an amino acid sequence shown in SEQ ID NO:    4;-   (A11) a protein comprising the amino acid sequence shown in SEQ ID    NO: 159;-   (A12) a protein comprising the amino acid sequence shown in SEQ ID    NO: 160;-   (A13) a protein comprising the amino acid sequence shown in SEQ ID    NO: 136;-   (A14) a protein comprising the amino acid sequence shown in SEQ ID    NO: 137;-   (A15) a protein comprising the amino acid sequence shown in SEQ ID    NO: 138;-   (A16) a protein comprising the amino acid sequence shown in SEQ ID    NO: 215;-   (A17) a protein comprising the amino acid sequence shown in SEQ ID    NO: 216;-   (A18) a protein comprising the amino acid sequence shown in SEQ ID    NO: 217;-   (A19) a protein comprising the amino acid sequence shown in SEQ ID    NO: 218;-   (A20) a protein comprising the amino acid sequence shown in SEQ ID    NO: 219;-   (A21) a protein comprising the amino acid sequence shown in SEQ ID    NO: 220;-   (A22) a protein comprising the amino acid sequence shown in SEQ ID    NO: 221;-   (A23) a protein comprising the amino acid sequence shown in SEQ ID    NO: 222;-   (A24) a protein comprising the amino acid sequence shown in SEQ ID    NO: 223;-   (A25) a protein comprising the amino acid sequence shown in SEQ ID    NO: 224;-   (A26) a protein comprising an ability to convert in the presence of    an electron transport system containing an electron donor, a    compound of formula (II) to a compound of formula (III), and    comprising an amino acid sequence having at least 80% sequence    identity with an amino acid sequence shown in any one of SEQ ID NO:    159, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 217,    SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221 or SEQ ID NO: 223 or    an amino acid sequence having at least 90% sequence identity with an    amino acid sequence shown in any one of SEQ ID NO: 160, SEQ ID NO:    215, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 222 or SEQ ID NO:    224; and-   (A27) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    90% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 160,    SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 215, SEQ    ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO: 219, SEQ ID    NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 or SEQ ID    NO: 224.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the annealing site of the PCR primers utilized to obtainthe present invention DNA (A1) and the present invention DNA (B1). Eachof the numbers refers to the SEQ ID number showing the nucleotidesequence of the primers. The arrows show the annealing sites of theoligonucleotide primers having the nucleotide sequence shown with theSEQ ID number thereof and the extention direction of the DNA polymerasereaction from the primers. The dotted lines represent the DNA amplifiedby the PCR utilizing the primers. The thick line represents the regionadjacent to the DNA insertion site of the vector utilized to produce thechromosomal DNA library.

FIG. 2 shows the annealing site of the PCR primers utilized to obtainthe present invention DNA (A2) and the present invention DNA (B2). Eachof the numbers refers to the SEQ ID number showing the nucleotidesequence of the primers. The arrows show the annealing sites of theoligonucleotide primers having the nucleotide sequence shown with theSEQ ID number thereof and the extention direction of the DNA polymerasereaction from the primers. The dotted lines represent the DNA amplifiedby the PCR utilizing the primers. The thick line represents the regionadjacent to the DNA insertion site of the vector utilized to produce thechromosomal DNA library.

FIG. 3 shows the annealing site of the PCR primers utilized to obtainthe present invention DNA (A4) and the present invention DNA (B4). Eachof the numbers refers to the SEQ ID number showing the nucleotidesequence of the primers. The arrows show the annealing sites of theoligonucleotide primers having the nucleotide sequence shown with theSEQ ID number thereof and the extention direction of the DNA polymerasereaction from the primers. The dotted lines represent the DNA amplifiedby the PCR utilizing the primers. The thick line represents the regionadjacent to the DNA insertion site of the vector utilized to produce thechromosomal DNA library. However, the oligonucleotide primer representedby 57, is a primer which anneals to the region adjacent to the DNAinsertion site of the vector utilized to produce the chromosomal DNAlibrary, and fails to anneal with the present invention DNA (A4).

FIG. 4 shows the restriction map of the plasmid pKSN2.

FIG. 5 shows the restriction map of the plasmid pCRrSt12.

FIG. 6 shows the restriction map of the plasmid pCR657ET.

FIG. 7 shows the restriction map of the plasmid pCR657FET.

FIG. 8 shows the restriction map of the plasmid pCR657Bs.

FIG. 9 shows the restriction map of the plasmid pCR657FBs.

FIG. 10 shows the restriction map of the plasmid pUCrSt12.

FIG. 11 shows the restriction map of the plasmid pUCrSt657.

FIG. 12 shows the restriction map of the plasmid pUCrSt657F.

FIG. 13 shows the restriction map of the plasmid pUCCR16G6-p/t.

FIG. 14 shows the structure of the linker NotI-EcoRI produced byannealing the oligonucleotide consisting of the nucleotide sequenceshown in SEQ ID NO: 89 and the oligonucleotide consisting of thenucleotide sequence shown in SEQ ID NO: 90.

FIG. 15 shows the restriction map of the plasmid pUCCR16G6-p/tΔ.

FIG. 16 shows the structure of the linker HindIII-NotI produced byannealing the oligonucleotide consisting of the nucleotide sequenceshown in SEQ ID NO: 91 and the oligonucleotide consisting of thenucleotide sequence shown in SEQ ID NO: 92.

FIG. 17 shows the restriction map of the plasmid pNdG6-ΔT.

FIG. 18 shows the restriction map of the plasmid pSUM-NdG6-rSt657.

FIG. 19 shows the restriction map of the plasmid pSUM-NdG6-rSt657F.

FIG. 20 shows the restriction map of the plasmid pKFrSt12.

FIG. 21 shows the restriction map of the plasmid pKFrSt12-657.

FIG. 22 shows the restriction map of the plasmid pKFrSt12-657F.

FIG. 23 shows the restriction map of the plasmid pSUM-NdG6-rSt12-657.

FIG. 24 shows the restriction map of the plasmid pSUM-NdG6-rSt12-657F.

FIG. 25 shows the structure of the linker HindIII-NotI-EcoRI produced byannealing the oligonucleotide consisting of the nucleotide sequenceshown in SEQ ID NO: 98 and the oligonucleotide consisting of thenucleotide sequence shown in SEQ ID NO: 99.

FIG. 26 shows the restriction map of te plasmid pBI121S.

FIG. 27 shows the restriction map of the plasmid pBI-NdG6-rSt657.

FIG. 28 shows the restriction map of the plasmid pBI-NdG6-rSt-657F.

FIG. 29 shows the restriction map of the plasmid pBI-NdG6-rSt12-657.

FIG. 30 shows the restriction map of the plasmid pBI-NdG6-rSt12-657F.

FIG. 31 shows the restriction map of the plasmid pCR923Sp.

FIG. 32 shows the restriction map of the plasmid pNdG6-rSt12.

FIG. 33 shows the restriction map of the plasmid pSUM-NdG6-rSt-923.

FIG. 34 shows the restriction map of the plasmid pKFrSt12-923.

FIG. 35 shows the restriction map of the plasmid pSUM-NdG6-rSt12-923.

FIG. 36 shows the restriction map of the plasmid pBI-NdG6-rSt-923.

FIG. 37 shows the restriction map of the plasmid pBI-NdG6-rSt12-923.

FIG. 38 shows the restriction map of the plasmid pCR671ET.

FIG. 39 shows the restriction map of the plasmid pCR671Bs.

FIG. 40 shows the restriction map of the plasmid pUCrSt671.

FIG. 41 shows the restriction map of the plasmid pSUM-NdG6-rSt671.

FIG. 42 shows the restriction map of the plasmid pKFrSt12-671.

FIG. 43 shows the restriction map of the plasmid pSUM-NdG6-rSt12-671.

FIG. 44 shows the restriction map of the plasmid pBI-NdG6-rSt-671.

FIG. 45 shows the restriction map of the plasmid pBI-NdG6-rSt12-671.

FIG. 46 shows the results obtained by detecting with agarose gelelectrophoresis the DNA amplified by the PCR using as a primer theoligonucleotide having a partial nucleotide sequence of the presentinvention DNA(A). Lanes 1, 7, 8, 12, 19, 26, 27, 32, 37, 42 and 47represent the electrophoresis of a DNA marker (φ174/HaeIII digest). Theother lanes represent the electrophoresis of the samples shown in Tables20 and 21.

FIG. 47 shows the structure of the linker produced by annealing theoligonucleotide consisting of the nucleotide sequence shown in SEQ IDNO: 134 and the oligonucleotide consisting of the nucleotide sequenceshown in SEQ ID NO: 135.

FIG. 48 shows the restriction map of the plasmid pUCrSt657soy.

FIG. 49 shows the restriction map of the plasmid pSUM-NdG6-rSt-657soy.

FIG. 50 shows the restriction map of the plasmid pKFrSt12-657soy.

FIG. 51 shows the restriction map of the plasmid pSUM-NdG6-rSt12657soy.

FIG. 52 shows the restriction map of the plasmid pBI-NdG6-rSt-657soy.

FIG. 53 shows the restriction map of the plasmid pBI-NdG6-rSt12-657soy.

FIG. 54 shows the restriction map of the plasmid pUCrSt1584soy.

FIG. 55 shows the restriction map of the plasmid pSUM-NdG6-rSt-1584soy.

FIG. 56 shows the restriction map of the plasmid pKFrSt12-1584soy.

FIG. 57 shows the restriction map of the plasmidpSUM-NdG6-rSt12-1584soy.

FIG. 58 shows the restriction map of the plasmid pBI-NdG6-rSt-1584soy.

FIG. 59 shows the restriction map of the plasmid pBI-NdG6-rSt12-1584soy.

FIG. 60 shows the restriction map of the plasmid pUCrSt1609soy.

FIG. 61 shows the restriction map of the plasmid pSUM-NdG6-rSt-1609soy.

FIG. 62 shows the structure of the linker EcoT22I-12aa-EcoT22I producedby annealing the oligonucleotide consisting of the nucleotide sequenceshown in SEQ ID NO: 402 and the oligonucleotide consisting of thenucleotide sequence shown in SEQ ID NO: 403.

FIG. 63 shows the restriction map of the plasmid pUCrSt12-1609soy.

FIG. 64 shows the restriction map of the plasmidpSUM-NdG6-rSt12-1609soy.

FIG. 65 shows the restriction map of the plasmid pBI-NdG6-rSt-1609soy.

FIG. 66 shows the restriction map of the plasmid pBI-NdG6-rSt12-1609soy.

The abbreviations described in the above figures are explained below.

-   DNA A1: the present invention DNA (A1)-   DNA A2: the present invention DNA (A2)-   DNA A3: the present invention DNA (A3)-   DNA A4: the present invention DNA (A4)-   DNA B1: the present invention DNA (B1)-   DNA B2; the present invention DNA (B2)-   DNA B4: the present invention DNA (B4)-   DNA A1S: the present invention DNA (A1)S-   DNA A23S: the present invention DNA (A23)S-   DNA A25S: the present invention DNA (A25)S-   tac p: tac promoter-   mB t; mB terminator-   ColE1 ori: the replication origin of plasmid ColE1-   Amp^(r): the ampicillin resistance gene-   RuBPCssCTP: the nucleotide sequence encoding the chloroplast transit    peptide of the small subunit of ribulose-1,5-bisphosphate    carboxylase of soybean (cv. Jack).-   12aa: the nucleotide sequence encoding the 12 amino acids of a    mature protein, following the chloroplast transit peptide of the    small subunit of ribulose-1,5-bisphosphate carboxylase of soybean    (cv Jack).-   Km^(r): kanamycin resistance gene-   F1 ori; replication origin of plasmid F1-   CR16G6p: CR16G6 promoter-   CR16t: CR16 terminator-   CR16tΔ: DNA in which the nucleotide sequence downstream of    restriction site of the restriction enzyme Scar is removed from the    CR16 terminator-   CR16G6pΔ: DNA in which the nucleotide sequence upstream of    restriction site of the restriction enzyme NdeI is removed from the    CR16G6 terminator-   NOSp: promoter of the nopaline synthase gene-   NPTII: kanamycin resistance gene-   NOSt: terminator of nopaline synthase gene-   GUS: β-glucuronidase gene-   RB: the right border sequence of T-DNA-   LB: the left border sequence of T-DNA-   NdeI, HindIII, BspHI, EcoRI, BamHI, EcoT221, SphI, KpnI, SacI,    BglII, NotI, ScaI: the restriction sites of the respective    restriction enzyme.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is explained in detail below.

The herbicide metabolizing protein selected from the following proteingroup (hereinafter, sometimes referred to as “the present inventionprotein (A)”) has the ability to convert the compound of formula (II)hereinafter, sometimes referred to as “compound (II)”) to the compoundof formula (III) (hereinafter, sometimes referred to as “compound(III)”).

Protein Group

-   (A1) a protein comprising the amino acid sequence shown in SEQ ID    NO: 1;-   (A2) a protein comprising the amino acid sequence shown in SEQ ID    NO: 2;-   (A3) a protein comprising the amino acid sequence shown in SEQ ID    NO: 3;-   (A4) a protein comprising the amino acid sequence shown in SEQ ID    NO: 108;-   (A5) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor a compound of    formula (II) to a compound of formula (III) and comprising an amino    acid sequence having at least 80% sequence identity with an amino    acid sequence shown in any one of SEQ ID NO; 1, SEQ ID NO: 2, SEQ ID    NO: 3 or SEQ ID NO: 108;-   (A6) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor a compound of    formula (II) to a compound of formula (III), and comprising an amino    acid sequence encoded by a nucleotide sequence having at least 80%    sequence identity with a nucleotide sequence encoding an amino acid    sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO 3    or SEQ ID NO: 108;-   (A11) a protein comprising the amino acid sequence shown in SEQ ID    NO: 159;-   (A12) a protein comprising the amino acid sequence shown in SEQ ID    NO: 160;-   (A13) a protein comprising the amino acid sequence shown in SEQ ID    NO: 136;-   (A14) a protein comprising the amino acid sequence shown in SEQ ID    NO: 137;-   (A15) a protein comprising the amino acid sequence shown in SEQ ID    NO: 138;-   (A16) a protein comprising the amino acid sequence shown in SEQ ID    NO: 215;-   (A17) a protein comprising the amino acid sequence shown in SEQ ID    NO: 216;-   (A18) a protein comprising the amino acid sequence shown in SEQ ID    NO 217;-   (A19) a protein comprising the amino acid sequence shown in SEQ ID    NO: 218;-   (A20) a protein comprising the amino acid sequence shown in SEQ ID    NO: 219;-   (A21) a protein comprising the amino acid sequence shown in SEQ ID    NO: 220;-   (A22) a protein comprising the amino acid sequence shown in SEQ ID    NO: 221;-   (A23) a protein comprising the amino acid sequence shown in SEQ ID    NO: 222;-   (A24) a protein comprising the amino acid sequence shown in SEQ ID    NO: 223;-   (A25) a protein comprising the amino acid sequence shown in SEQ ID    NO: 224;-   (A26) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor a compound of    formula (II) to a compound of formula (III), and comprising an amino    acid sequence having at least 80% sequence identity with an amino    acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 136,    SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219, SEQ    ID NO: 220, SEQ ID NO: 221 or SEQ ID NO: 223 or an amino acid    sequence having at least 90% sequence identity with an amino acid    sequence shown in any one of SEQ ID NO: 160, SEQ ID NO: 215, SEQ ID    NO: 216, SEQ ID NO: 218, SEQ ID NO: 222 or SEQ ID NO: 224;-   (A27) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor a compound of    formula (II) to a compound of formula (III), and comprising an amino    acid sequence encoded by a nucleotide sequence having at least 90%    sequence identity with a nucleotide sequence encoding an amino acid    sequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID    NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 215, SEQ ID NO,    216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO: 219, SEQ ID NO: 220,    SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 or SEQ ID NO: 224;    and-   (A28) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a DNA amplifiable by a polymerase    chain reaction with a primer comprising the nucleotide sequence    shown in any one of SEQ ID NOs: 124 to 128, a primer comprising the    nucleotide sequence shown in SEQ ID NO: 129 and as a template a    chromosomal DNA of Streptomyces phaeochromogenes, Streptomyces    testaceus, Streptomyces achromogenes, Streptomyces griseofuscus,    Streptomyces thermocoerulescens, Streptomyces nogalater,    Streptomyces tsusimaensis, Streptomyces glomerochromogenes,    Streptomyces olivochromogenes, Streptomyces ornatus, Streptomyces    griseus, Streptomyces lanatus, Streptomyces misawanensis,    Streptomyces pallidus, Streptomyces roseorubens, Streptomyces    rutgersensis, Streptomyces steffisburgensis or Saccharopolyspora    taberi.

As specific examples of the present invention protein (A), there ismentioned:

-   -   a protein comprising the amino acid sequence shown in SEQ [D NO:        1 (hereinafter, sometimes referred to as “present invention        protein (A1)”);    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        2 (hereinafter, sometimes referred to as “present invention        protein (A2)”);    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        3 (hereinafter, sometimes referred to as “present invention        protein (A3)”);    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        108 (hereinafter, sometimes referred to as “present invention        protein (A4)”);    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        159 (hereinafter, sometimes referred to as “present invention        protein (A11)”);    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        160 (hereinafter, sometimes referred to as “present invention        protein (A12)”);    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        136 (hereinafter, sometimes referred to as “present invention        protein (A13)”);    -   a protein comprising the amino acid sequence shown in SEQ ID NO;        137 (hereinafter, sometimes referred to as “present invention        protein (A14)”);    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        138 (hereinafter, sometimes referred to as “present invention        protein (A15)”);    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        215 (hereinafter, sometimes referred to as “present invention        protein (A16)”);    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        216 (hereinafter, sometimes referred to as “present invention        protein (A17)”);    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        217 (hereinafter, sometimes referred to as “present invention        protein (A18)”);    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        218 (hereinafter, sometimes referred to as “present invention        protein (A19)”);    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        219 (hereinafter, sometimes referred to as “present invention        protein (A20)”);    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        220 (hereinafter, sometimes referred to as “present invention        protein (A21)”);    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        221 (hereinafter, sometimes referred to as “present invention        protein (A22)”);    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        222 (hereinafter, sometimes referred to as “present invention        protein (A23)”);    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        223 (hereinafter, sometimes referred to as “present invention        protein (A24)”); and    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        224 (hereinafter, sometimes referred to as “present invention        protein (A25)”).

For example, by reacting the PPO inhibitory-type herbicidal compound offormula (I) (hereinafter, sometimes referred to as “compound (I)”) withthe present invention protein (A), it is capable to convert the compoundto a compound with lower herbicidal activity.

Further, in treatment to convert compound (I) to a compound of a lowerherbicidal activity, there can also be utilized a herbicide metabolizingprotein selected from the following group (hereinafter, sometimesreferred to as “present protein (A)”):

Protein Group

-   (A1) a protein comprising the amino acid sequence shown in SEQ ID    NO: 1;-   (A2) a protein comprising the amino acid sequence shown in SEQ ID    NO: 2;-   (A3) a protein comprising the amino acid sequence shown in SEQ ID    NO: 3;-   (A4) a protein comprising the amino acid sequence shown in SEQ ID    NO: 108;-   (A5) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor a compound of    formula (II) to a compound of formula (III), and comprising an amino    acid sequence having at least 80% sequence identity with an amino    acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID    NO: 3 or SEQ ID NO: 108;-   (A6) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor a compound of    formula (II) to a compound of formula (III), and comprising an amino    acid sequence encoded by a nucleotide sequence having at least 80%    sequence identity with a nucleotide sequence encoding an amino acid    sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:    3 or SEQ ID NO: 108; (A7) a protein having the ability to convert in    the presence of an electron transport system containing an electron    donor a compound of formula (II) to a compound of formula (III), and    comprising an amino acid sequence encoded by a DNA that hybridizes,    under stringent conditions, to a DNA comprising a nucleotide    sequence encoding an amino acid sequence shown in any one of SEQ ID    NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ LD NO: 108;-   (A8) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor a compound of    formula (II) to a compound of formula (III), and comprising an amino    acid sequence encoded by a DNA amplifiable by a polymerase chain    reaction with a primer comprising a nucleotide sequence shown in SEQ    ID NO: 129, a primer comprising a nucleotide sequence shown in any    one of SEQ ID NOs: 124 to 128, and as a template a chromosome of a    microorganism belonging to Streptomyces or Saccharopolyspora;-   (A9) a protein comprising an amino acid sequence shown in SEQ ID NO:    4,-   (A11) a protein comprising the amino acid sequence shown in SEQ ID    NO: 159;-   (A12) a protein comprising the amino acid sequence shown in SEQ ID    NO: 160;-   (A13) a protein comprising the amino acid sequence shown in SEQ ID    NO: 136;-   (A14) a protein comprising the amino acid sequence shown in SEQ ID    NO: 137;-   (A15) a protein comprising the amino acid sequence shown in SEQ ID    NO: 138;-   (A16) a protein comprising the amino acid sequence shown in SEQ ID    NO; 215;-   (A17) a protein comprising the amino acid sequence shown in SEQ ID    NO: 216;-   (A18) a protein comprising the amino acid sequence shown in SEQ ID    NO: 217;-   (A19) a protein comprising the amino acid sequence shown in SEQ ID    NO: 218;-   (A20) a protein comprising the amino acid sequence shown in SEQ ID    NO: 219;-   (A21) a protein comprising the amino acid sequence shown in SEQ ID    NO: 220;-   (A22) a protein comprising the amino acid sequence shown in SEQ ID    NO: 221;-   (A23) a protein comprising the amino acid sequence shown in SEQ ID    NO: 222;-   (A24) a protein comprising the amino acid sequence shown in SEQ ID    NO: 223;-   (A25) a protein comprising the amino acid sequence shown in SEQ ID    NO: 224;-   (A26) a protein having an ability to convert in the presence of an    electron transport system containing an electron donor a compound of    formula (II) to a compound of formula (III), and comprising an amino    acid sequence having at least 80% sequence identity with an amino    acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 136,    SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219, SEQ    ID NO: 220, SEQ ID NO: 221 or SEQ ID NO: 223 or an amino acid    sequence having at least 90% sequence identity with an amino acid    sequence shown in any one of SEQ ID NO: 160, SEQ ID NO: 215, SEQ ID    NO: 216, SEQ ID NO: 218, SEQ ID NO: 222 or SEQ ID NO: 224; and-   (A27) a protein having the ability to convert in the presence of an    electron transport system containing an electron donor, a compound    of formula (II) to a compound of formula (III), and comprising an    amino acid sequence encoded by a nucleotide sequence having at least    90% sequence identity with a nucleotide sequence encoding an amino    acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 160,    SEQ ID NO; 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 215, SEQ    ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO: 219, SEQ ID    NO: 220, SEQ ID NO: 221, SEQ ID NO 222, SEQ ID NO: 223 or SEQ ID NO:    224.

As examples of the present protein (A), there can be mentioned thepresent invention protein A, described above. Further, as otherexamples, there can be mentioned

-   -   a protein comprising the amino acid sequence shown in SEQ ID NO:        4 (hereinafter, sometimes referred to as “present protein (A9)”)        and    -   a protein comprising the amino acid sequence shown in SEQ ID NO:        5 (hereinafter, sometimes referred to as “present protein        (A10)”).

In the amino acid sequence of the protein shown in (A5), (A6), (A7),(A8), (A26), (A27) or (A28) in the above protein groups, the differenceswhich may be observed from the amino acid sequences shown in SEQ ID NO:1, 2, 3, 108, 159, 160, 136, 137, 138, 215, 216, 217, 218, 219, 220,221, 222, 223 or 224, are such as deletion, substitution, and additionof certain amino acids. Such differences include, for example, thedeletion from the processing which the above protein comprising theamino acid sequence shown in SEQ ID NO: 1, 2, 3, 108, 159, 160, 136,137, 138, 215, 216, 217, 218, 219, 220, 221, 222, 223 or 224 receiveswithin the cell. Further, there are included a polymorphic variationwhich occurs naturally resulting from the difference by such as thespecies, individual or the like of the organism from which the proteinis derived; amino acid deletions, substitutions, and additions arisingfrom genetic mutations artificially introduced by such as asite-directed mutagenesis method, a random mutagenesis method, amutagenic treatment and the like.

The number of amino acids undergoing such deletions, substitutions andadditions may be within the range in which the present protein (A) candevelop the ability to convert compound (II) to compound (III). Further,as a substitution of the amino acid, there can be mentioned, forexample, substitutions to an amino acid which is similar inhydrophobicity, charge, pK, stereo-structural feature, or the like. Assuch substitutions, specifically for example, there are mentionedsubstitutions within the groups of: (1.) glycine and alanine; (2.)valine, isoleucine and leucine; (3.) aspartic acid, glutamic acid,asparagine and glutamine; (4.) serine and threonine; (5.) lysine andarginine; (6.) phenylalanine and tyrosine; and the like.

Further, in the present protein (A), it is preferable that the cysteinepresent at the position aligning to the cysteine of amino acid number357 in the amino acid sequence shown in SEQ ID NO: 1 is conserved (notundergo a deletion or substitution): examples of such cysteine includethe cysteine shown at amino acid number 350 in the amino acid sequenceshown in SEQ ID NO: 2, the cysteine shown at amino acid number 344 inthe amino acid sequence shown in SEQ ID NO 3, the cysteine shown atamino acid number 360 in the amino acid sequence shown in SEQ ID NO:108; the cysteine shown at amino acid number 359 in the amino acidsequence shown in SEQ ID NO: 4, the cysteine shown at amino acid number355 in the amino acid sequence shown in SEQ ID NO: 5, the cysteine shownat amino acid number 358 in the amino acid sequence shown in SEQ ID NO:159, the cysteine shown at amino acid number 374 in the amino acidsequence shown in SEQ ID NO: 160, the cysteine shown at amino acidnumber 351 in the amino acid sequence shown in SEQ ID NO: 136, thecysteine shown at amino acid number 358 in the amino acid sequence shownin SEQ ID NO: 137, the cysteine shown at amino acid number 358 in theamino acid sequence shown in SEQ ID NO: 138, the cysteine shown at aminoacid number 347 in the amino acid sequence shown in SEQ ID NO: 222, thecysteine shown at amino acid number 347 in the amino acid sequence shownin SEQ ID NO: 224 and the like.

As methods of artificially causing such amino acid deletions, additionsor substitutions (hereinafter, sometimes, collectively referred to as“amino acid modification”), for example, there is mentioned a methodcomprising the steps of carrying out site-directed mutagenesis on theDNA encoding an amino acid sequence shown in any one of SEQ ID NO: 1, 2,3, 108, 159, 160, 136, 137, 138, 215, 216, 217, 218, 219, 220, 221, 222,223 or 224, and then allowing the expression of such DNA by aconventional method. As the site-directed mutagenesis method, forexample, there is mentioned a method which utilizes amber mutations(Gapped Duplex method, Nucleic Acids Res., 12, 9441-9456 (1984)), amethod by PCR utilizing primers for introducing a mutation and the like.Further, as methods of artificially modifying amino acids, for example,there is mentioned a method comprising the steps of carrying out randommutagenesis on the DNA encoding any one of the amino acid sequencesshown in SEQ ID NO: 1, 2, 3, 108, 159, 160, 136, 137, 138, 215, 216,217, 218, 219, 220, 221, 222, 223 or 224 and then allowing theexpression of such DNA by a conventional method. As the randommutagenesis method, for example, there is mentioned method of conductingPCR by utilizing the DNA encoding any one of the above amino acidsequences as a template an by utilizing a primer pair which can amplifythe full length of each of the DNA, under the condition in which theconcentration of each of dATP, dTTP, dGTP and dCTP, utilized as asubstrate, are different than usual or under the condition in which theconcentration of Mg²⁺ that promotes the polymerase reaction is increasedto wore than usual. As such methods of PCR, for example, there ismentioned the method described in Method in Molecular Biology, (31),1994, 97-112. Further, there may be mentioned the method described inPCT patent publication WO 00/09682.

In the present invention, “sequence identity” refers to the homology andidentity between two nucleotide sequences or two amino acid sequences.Such “sequence identity” may be determined by comparing the twosequences, each aligned in an optimal state, over the whole region ofthe test sequences. As such, additions or deletions (for example, gaps)can be utilized in the optimal alignment of the test nucleic acidsequences or amino acid sequences. Such sequence identity can becalculated through the step of producing the alignment conducted by ahomology analysis using a program such as FASTA (Pearson & Lipman, Proc.Natl. Acad. Sci. USA, 4, 2444-2448 (1988)), BLAST (Altschul et al.,Journal of Molecular Biology, 215, 463-410 (1990)), CLUSTAL W (Thompson,Higgins & Gibson, Nucleic Acid Research, 22, 4673-4680 (1994a)) and thelike. Such programs, for example, can be typically utilized on thewebpage (http://www.ddbj.nig.ac.jp) of the DNA Data Bank of Japan (theinternational databank operated within the Center for InformationBiology and DNA Data Bank of Japan). Further, the sequence identity maybe determined by utilizing a commercially available sequence analysissoftware. Specifically for example, it can be calculated by producing analignment conducted by a homology analysis by the Lipman-Pearson method(Lipman, D. J. and Pearson, W. R., Science, 227, 1435-1441, (1985))utilizing GENETYX-WIN Ver.5 (Software Development Company, Ltd.).

As the “stringent condition” described in (A7), there can be mentioned,for example, the conditions under which a hybrid is formed at 45° C. ina solution containing 6×SSC (let the solution containing 1.5 M NaCl and0.15 M trisodium citrate be 10×SC) and then the hybrid is washed at 50°C. with 2×SSC (Molecular Biology, John Wiley & Sons, N.Y. (1989),6.31-6.3.6) in a hybridization conducted according to the conventionalmethod described in such as Sambrook, J., Frisch, E. F., and Maniatis,T.; Molecular Cloning 2nd edition, Cold Spring Harbor Press. The saltconcentration in the washing step can be selected, for example, from theconditions of 2×SSC (low stringency condition) to the conditions of0.2×SSC (high stringency conditions). A temperature in the washing stepcan be selected, for example, from room temperature (low stringencycondition) to 65° C. (high stringency condition). Alternatively, both ofthe salt concentration and temperature may be changed.

As a DNA which “hybridizes, under stringent conditions, to a DNAcomprising a nucleotide sequence encoding an amino acid sequence shownin any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:108”, specifically for example, there can be mentioned a DNA comprisinga nucleotide sequence encoding an amino acid sequence shown in any oneof SEQ ID NO: 1, 2, 3, 4, 5, 108, 159, 160, 136, 137, 138, 215, 216,217, 218, 219, 220, 221, 222, 223 or 224, a DNA comprising a nucleotidesequence shown in any one of SEQ ID NO: 6, 7, 8, 78, 84, 109, 139, 140,141, 142, 143, 225, 226, 227, 228, 229, 230, 231, 232, 233 or 234, andthe like. There can also be mentioned DNA comprising a nucleotidesequence having at least about 60% identity to a nucleotide sequenceshown in any one of SEQ ID NO: 6, 7, 8, 78, 84, 109, 139, 140, 141, 142,143, 225, 226, 227, 228, 229, 230, 231, 232, 233 or 234.

The molecular weight of the present protein (A) is about 30,000 to60,000 and is typically about 40,000 to 50,000 (comparable to, forexample, a protein consisting of the amino acid sequence shown in anyone of SEQ ID NO: 1, 2, 3, 108, 159, 160, 136, 137, 138, 215, 216, 217,218, 219, 220, 221, 222, 223 or 224), as the molecular weight identifiedby a sodium dodecyl sulfate-polyacrylamide gel electrophoresis(hereinafter, referred to as “SDS-PAGE”). Further, the present protein(A), as long as the ability to convert compound (II) to compound (II) isnot eliminated, can be utilized as a protein to which amino acidsequence is added upstream to its amino terminus or downstream to itscarboxy terminus.

As the marker of the ability of the present protein (A) to metabolizethe PPO inhibitory-type herbicidal compound of formula (I), there can bementioned the ability to convert compound (II) to compound (III). Suchability, for example, can be confirmed by reacting compound (II) withthe present protein (A) in the presence of an electron transport systemcontaining an electron donor such as coenzyme NADPH and by detecting theproduced compound (III).

The “electron transport system containing an electron donor” refers to asystem in which a redox chain reaction occurs and an electron istransferred from the electron donor to the present protein (A). As theelectron donor, for example, there is mentioned coenzymes NADPH, NADHand the like. For example, as proteins which may constitute the electrontransport system from NADPH to the present protein (A), there ismentioned ferredoxin and ferredoxin-NADP⁺ reductase, NADPH-cytochromeP-450 reducase, and the like.

To confirm the ability of converting compound (II) to compound (III),for example, a reaction solution of about pH 7, comprising the presentprotein (A), B NADPH, ferredoxin, ferredoxin-NADP⁺ reductase andcompound (II) labeled with a radioisotope, is incubated at about 30° C.for about 10 minutes to 1 hour. Subsequently, after making the reactionsolution acidic by adding hydrochloric acid, it is extracted with ethylacetate. After subjecting the recovered ethyl acetate layer to thinlayered chromatography (hereinafter referred to as “TLC”),autoradiography is conducted and the ability to convert compound (II) tocompound (III) can be confirmed by detecting the labeled compound (III).

To prepare the present protein (A), for example, first, the DNA encodingthe present protein (A) (hereinafter, sometimes collectively referred toas “present DNA (A)”) is obtained according to the conventional geneticengineering methods (for example, the methods described in Sambrook, J.,Frisch, E. F., Maniatis, T.; Molecular Cloning 2nd Edition, Cold SpringHarbor Laboratory press).

As examples of the present DNA (A), there can be mentioned a DNAencoding the present invention protein (A) (hereinafter, sometimesreferred to as “present invention DNA (A)”). As specific examples of thepresent invention DNA (A), there can be mentioned:

-   -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 1 (hereinafter, sometimes referred to as        “present invention DNA (A1)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 2 (hereinafter, sometimes referred to as        “present invention DNA (A2)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 3 (hereinafter, sometimes referred to as        “present invention DNA (A3)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 108 (hereinafter, sometimes referred to as        “present invention DNA (A4)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 159 (hereinafter, sometimes referred to as        “present invention DNA (A11)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 160 (hereinafter, sometimes referred to as        “present invention DNA (A12)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 136 (hereinafter, sometimes referred to as        “present invention DNA (A13)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 137 (hereinafter, sometimes referred to as        “present invention DNA (A14)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 138 (hereinafter, sometimes referred to as        “present invention DNA (A15)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 215 (hereinafter, sometimes referred to as        “present invention DNA (A16)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 216 (hereinafter, sometimes referred to as        “present invention DNA (A17)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 217 (hereinafter, sometimes referred to as        “present invention DNA (A18)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 218 (hereinafter, sometimes referred to as        “present invention DNA (A19)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 219 (hereinafter, sometimes referred to as        “present invention DNA (A20)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 220 (hereinafter, sometimes referred to as        “present invention DNA (A21)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 221 (hereinafter, sometimes referred to as        “present invention DNA (A22)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 222 (hereinafter, sometimes referred to as        “present invention DNA (A23)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 223 (hereinafter, sometimes referred to as        “present invention DNA (A24)”);    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 224 (hereinafter, sometimes referred to as        “present invention DNA (A25)”); and the like.

Further as more specific examples of the present invention DNA (A),there can be mentioned:

-   -   a DNA comprising the nucleotide sequence shown in SEQ ID NO: 6;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO: 9;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO: 7;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO: 10;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO: 8;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO: 11;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        109;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        110;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        139;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        144;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO,        140;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        145;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        141;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        146;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        142;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        147;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        143;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        148;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        225;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        235;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        226;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        236;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        227;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        237;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        228;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        238;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        229;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        239;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        230;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        240;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        231;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        241;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        232;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        242;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO,        233;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        243;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        234;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        244;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        214;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        368;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO:        393;    -   a DNA encoding a protein having an ability to convert in the        presence of an electron transport system containing an electron        donor a compound of formula (II) to a compound of formula (III),        and having at least 80% sequence identity with a nucleotide        sequence shown in any one of SEQ ID NO: 6, 7, 8 or 109;    -   a DNA encoding a protein having an ability to convert in the        presence of an electron transport system containing an electron        donor a compound of formula (II) to a compound of formula (III),        and having at least 90% sequence identity with a nucleotide        sequences shown in any one of SEQ ID NO: 139, 140, 141, 142,        143, 225, 226, 227, 228, 229, 230, 231, 232, 233 or 234; and the        like.

Further, as examples of the present DNA (A), other than the presentinvention DNA (A) above, there is mentioned:

-   -   a DNA comprising the nucleotide sequence encoding a protein        comprising the amino acid sequence shown in SEQ ID NO: 4        (hereinafter, sometimes referred to as “present DNA (A9)”);    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO: 78;    -   a DNA comprising the nucleotide sequence encoding a protein        comprising the amino acid sequence shown in SEQ ID NO: 5        (hereinafter, sometimes referred to as “present DNA (A10)”);    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO: 84;    -   a DNA comprising the nucleotide sequence shown in SEQ ID NO: 85;        and the like.

The present DNA(A), for example, may be a DNA cloned from nature and maybe a DNA in which a deletion, substitution or addition of nucleotide(s)has been introduced to the DNA cloned from nature by such as asite-directed mutagenesis method, a random mutagenesis method, and maybe an artificially synthesized DNA. Subsequently, the present protein(A) can be produced or obtained by expressing the obtained present DNA(A) according to the conventional genetic engineering methods. In suchways, the present protein (A) can be prepared.

The present DNA (A) can be prepared, for example, by the followingmethods. First, chromosomal DNA is prepared by conventional geneticengineering methods, such as those described in Molecular Cloning: ALaboratory Manual 2nd edition (1989), Cold Spring Harbor LaboratoryPress; and Current Protocols in Molecular Biology (1987), John Wiley &Sons, Incorporated, from microorganisms belonging to Streptomyces, suchas Streptomyces phaeochromogenes, Streptomyces testaceus, Streptomycesachromogenes, Streptomyces griseolus, Streptomyces carbophilus,Streptomyces griseofuscus, Streptomyces thermocoerulescens, Streptomycesnogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes,Streptomyces olivochromogenes, Streptomyces ornatus, Streptomycesgriseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomycespallidus, Streptomyces roseorubens, Streptomyces rutgersensis andStreptomyces steffisburgensis, and more specifically, Streptomycesphaeochromogenes IFO12898, Streptomyces testaceus ATCC21469,Streptomyces achromogenes IFO 12735, Streptomyces griseolus ATCC 11796,Streptomyces carbophilus SANK62585, Streptomyces griseofuscus IFO12870t, Streptomyces thermocoerulescens IFO 14273t, Streptomycesnogalater IFO 13445, Streptomyces tsusimaensis IFO 13782, Streptomycesglomerochromogenes IFO 13673t, Streptomyces olivochromogenes IFO 12444,Streptomyces ornatus IFO 1306t Streptomyces griseus ATCC 10137,Streptomyces griseus IFO 13849T, Streptomyces lanatus IFO 12787T,Streptomyces misawanensis IFO 13855T, Streptomyces pallidus IFO 13434T,Streptomyces roseorubens IFO 13682T, Streptomyces rutgersensis IFO15875T and Streptomyces steffisburgensis IFO 13446T, and the like; ormicroorganisms belonging to Saccharopolyspora, such as Saccharopolysporataberi, more specifically, Saccharopolyspora taberi JCM 9383t and thelike. Next, after partial digestion of the chromosomal DNA with arestriction enzyme such as Sau3AI, a DNA of about 2 kb is recovered. Therecovered DNA is cloned into a vector according to the conventionalgenetic engineering methods described in “Molecular Cloning: ALaboratory Manual 2nd edition” (1989), Cold Spring Harbor LaboratoryPress; and “Current Protocols in Molecular Biology” (1987), John Wiley &Sons, Incorporated. As the vector, specifically for example, there canbe utilized pUC 119 (TaKaRa Shuzo Company), pTVA 118N (Takara ShuzoCompany), pBluescript II (Toyobo Company), pCR2.1-TOPO (Invitrogen),pTrc99A (Amersham Pharmacia Biotech Company), pKK331-1A (AmershamPharmacia Biotech Company), and the like. A chromosomal DNA library canbe obtained by extracting the plasmid from the obtained clone.

The present DNA (A) can be obtained by hybridizing a probe with theobtained chromosomal DNA library under the conditions described below,and by detecting and recovering the DNA which bound specifically withthe probe. The probe can be a DNA consisting of about at least 20nucleotides comprising the nucleotides sequence encoding an amino acidsequence shown in any one of SEQ ID NO: 1, 2, 3 or 108. As specificexamples of the DNA which can be utilized as probes, there is mentioneda DNA comprising a nucleic acid shown in any one of SEQ ID NO: 6, 7, 8or 109; a DNA comprising a partial nucleotide sequence of the nucleicacid sequence shown in any one of SEQ ID NO: 6, 7, 8 or 109; a DNAcomprising a nucleotide sequence complimentary to said partialnucleotide sequence; and the like.

The DNA utilized as the probe is labeled with a radioisotope,fluorescent coloring or the like. To label the DNA with a radioisotope,for example, there can be utilized the Random Labeling Kit of Boehringeror Takara Shuzo Company. Further, a DNA labeled with ³²P can be preparedby conducting PCR. The DNA to be utilized for the probe is utilized asthe template. The dCTP typically utilized in the PCR reaction solutionis exchanged with (α-³²P)dCTP. Further, when labeling the DNA withfluorescent coloring, for example, there can be utilized DIG-High PrimeDNA labeling and. Detection Starter Kit II (Roche Company).

A specific example of preparing the probe is explained next. Forexample, a DNA labeled with digoxigenin, comprising the full length ofthe nucleotide sequence shown in SEQ ID NO; 6 can be obtained byutilizing the chromosomal DNA prepared from Streptomycesphaeochromogenes IFO12898 as described above or a chromosomal DNAlibrary as a template, by utilizing as primers an oligonucleotideconsisting of the nucleotide sequence shown in SEQ ID NO: 93 and anoligonucleotide consisting of the nucleotide sequence shown in SEQ IDNO: 94, and by conducting PCR as described in the examples describedbelow with, for example, PCR DIG Probe Synthesis Kit (Roche DiagnosticsGmbH) according to the attached manual. Similarly, a DNA labeled withdigoxigenin, comprising the nucleotide sequence of from nucleotide 57 tonucleotide 730 shown in SEQ ID NO: 6 can be obtained by utilizing thechromosomal DNA prepared from Streptomyces phaeochromogenes IFO12898 asdescribed above or a chromosomal DNA library as the template As primers,the PCR is conducted with an oligonucleotide consisting of thenucleotide sequence shown in SEQ ID NO: 130 and an oligonucleotideconsisting of the nucleotide sequence shown in SEQ ID NO: 131. Further,a DNA labeled with digoxigenin, comprising the full length of thenucleotide sequence shown in SEQ ID NO: 7 can be obtained by utilizingthe chromosomal DNA prepared from Saccharopolyspora taberi JCM 9383t asdescribed above or a chromosomal DNA library as the template. Asprimers, the PCR is conducted with an oligonucleotide consisting of thenucleotide sequence shown in SEQ ID NO: 61 and an oligonucleotideconsisting of the nucleotide sequence shown in SEQ ID NO: 62. Further, aDNA labeled with digoxigenin, comprising the full length of thenucleotide sequence shown in SEQ ID NO: 8 can be obtained by utilizingthe chromosomal DNA prepared from Streptomyces testaceus ATCC21469 asdescribed above or a chromosomal DNA library as the template. Asprimers, the PCR is conducted with an oligonucleotide consisting of thenucleotide sequence shown in SEQ ID NO: 70 and an oligonucleotideconsisting of the nucleotide sequence shown in SEQ ID NO: 71. Further, aDNA labeled with digoxigenin, comprising the nucleotide sequence of fromnucleotide 21 to nucleotide 691 shown in SEQ ID NO: 8 can be obtained byutilizing the chromosomal DNA prepared from Streptomyces testaceusATCC21469 as described above or a chromosomal DNA library as thetemplate. As primers, the PCR is conducted with an oligonucleotideconsisting of the nucleotide sequence shown in SEQ ID NO: 132 and anoligonucleotide consisting of the nucleotide sequence shown in SEQ IDNO: 133.

The methods by which a probe is allowed to hybridize with thechromosomal DNA library may include colony hybridization and plaquehybridization, and an appropriate method may be selected, which iscompatible with the type of vector used in the library preparation. Whenthe utilized library is constructed with the use of plasmid vectors,colony hybridization is conducted. Specifically first, transformants areobtained by introducing the DNA of the library into microorganism inwhich the plasmid vector utilized to construct the library isreplicable. The obtained transformants are diluted and spread onto anagar plate and cultured until colonies appear. When a phage vector isutilized to construct the library, plaque hybridization is conducted.Specifically, first, the microorganism in which the phage vectorutilized to produce the library is replicable is mixed with the phage ofthe library, under the conditions in which infection is possible. Themixture is then further mixed with soft agar. This mixture is thenspread onto an agar plate. Subsequently, the mixture is cultured untilplaques appear.

Next, in the case of any one of the above hybridizations, a membrane isplaced on the surface of the agar plate in which the above culturing wasconducted and the colonies of the transformants or the phage particlesin the plaques are transferred to the membrane. After alkali treatmentof the membrane, there is a neutralization treatment. The DNA elutedfrom the transformants or the phage particles is then fixed onto themembrane. More specifically for example, in the event of plaquehybridization, the phage particles are absorbed onto the membrane byplacing a nitrocellulose membrane or a nylon membrane, specifically forexample, Hybond-N⁺ (Amersham Pharmacia Biotech Company) on the agarplate and waiting for 1 minute. The membrane is soaked in an alkalisolution (1.5M NaCl and 0.5N NaOH) for about 3 minutes to dissolve thephage particles and elute the phage DNA onto the membrane. The membraneis then soaked in neutralization solution (1.5M NaCl and 0.5M tris-HClbuffer pH7.5) for about 5 minutes. After washing the membrane in washingsolution (0.3M NaCl, 30 mM sodium citrate, 0.2M tris-HCl buffer pH7.5)for about 5 minutes, for example, the phage DNA is fixed onto themembrane by incubating about 80° C. for about 90 minutes in vacuo.

By utilizing the membrane prepared as such, hybridization is conductedwith the above DNA as a probe. Hybridization can be conducted, forexample, according to the description in “Molecular Cloning: ALaboratory Manual 2nd edition (1989)” Cold Spring Harbor LaboratoryPress, and the like.

While various temperature conditions and reagents are available forconducting hybridization, the membrane prepared as described above issoaked with and maintained for 1 hour to 4 hours at 42° C. to 65° C. ina prehybridization solution, which is prepared at a ratio of from 50 μlto 200 μl per 1 cm² of the membrane. The prehybridization solution, forexample, may contain 450 mM to 900 mM NaCl and 45 mM to 90 mM sodiumcitrate, contain sodium dodecyl sulfate (hereinafter, referred to as“SDS”) at a concentration of 0.1% to 1.0%, and contain denaturedunspecific DNA at a concentration of from 0 μg/ml to 200 μg/ml, and maysometimes contain albumin, phycol, and polyvinyl pyrrolidone, each at aconcentration of 0% to 0-2%. Subsequently, for example, the membrane issoaked with and maintained for 12 hours to 20 hours at 42° C. to 65° C.in a hybridization solution, which is prepared at a ratio of from 50 μlto 200 μl per 1 cm² of the membrane. The hybridization solution is, forexample, a mixture of the prehybridization solution, which may contain450 mM to 900 mM NaCl and 45 mM to 90 mM sodium citrate, contain SDS ata concentration of 0.1% to 1.0%, and contain denatured unspecific DNA ata concentration of from 0 μg/ml to 200 μg/ml, and may sometimes containalbumin, phycol, and polyvinyl pyrrolidone, each at a concentration of0% to 0.2%, with the probe obtained with the preparation methoddescribed above (in a relative amount of 1.0×10⁴ cpm to 2.0×10⁶ cpm per1 cm² of the membrane). Subsequently, the membrane is removed and a washof 5 minutes to 15 minutes is conducted about 2 to 4 times, utilizing awashing solution of 42° C. to 65° C. that contains 15 mM to 300 mM ofNaCl, 1.5 mM to 30 mM of sodium citrate and 0.1% to 1.0% of SDS.Further, after lightly rinsing with 2×SSC solution (300 mM NaCl and 30mM sodium citrate), the membrane is dried. By detecting the position ofthe probe on the membrane by subjecting the membrane to autoradiography,the position of the DNA hybridizing to the utilized probe on themembrane is identified. Alternatively, prehybridization andhybridization can be conducted with the use of a commercially availablehybridization kit, such as with the use of hybridization solutioncontained in the DIG-High Prime DNA Labeling and Detection Starter KitII (Roche). After hybridization, for example, the membrane is washedtwice for 5 minutes at room temperature in 2×SSC containing 0.1% SDS,followed by washing twice for 15 minutes at 65° C. in 0.5×SSC containing0.1% SDS. The positions of DNAs on the membrane hybridizing with theutilized probe are detected, by treating in turn the washed membranewith the detection solution contained in the kit and by detecting theposition of the probe on the membrane.

The clones corresponding to the positions of the detected DNAs on themembrane are identified on the original agar medium, and can be pickedup to isolate clones carrying those DNAs.

The present DNA (A) obtained according to the above can be cloned into avector according to conventional genetic engineering methods describedin “Molecular Cloning: A Laboratory Manual 2nd edition” (1989), ColdSpring Harbor Laboratory Press, “Current Protocols in Molecular Biology”(1987), John Wiley & Sons Incorporated, and the like. As the vector,specifically for example, there can be utilized pUCA119 (Takara ShuzoCompany), pTVA118N (Takara Shuzo Company), pBluescriptII (ToyoboCompany), pCR2.1-TOPO (Invitrogen Company), pTrc99A (Pharmacia Company),pKK331-1A (Pharmacia Company) and the like.

Further, the nucleotide sequence of the present DNA (A) obtainedaccording to the above description can be analyzed by the dideoxyterminator method described in F. Sanger, S. Nicklen, A. R. Coulson,Proceeding of National Academy of Science U.S.A. (1977) 74:5463-5467. Inthe sample preparation for the nucleotide sequence analysis, acommercially available reagent may be utilized, such as the ABI PRISMDye Terminator Cycle Sequencing Ready Reaction Kit of Perkin ElmerCompany.

The present DNA (A) can also be prepared as follows. The present DNA (A)can be amplified by conducting PCR. The PCR may utilize as a templatethe chromosomal DNA or chromosomal DNA library prepared as describedabove from microorganisms belonging to Streptomyces, such asStreptomyces phaeochromogenes, Streptomyces testaceus, Streptomycesachromogenes, Streptomyces griseolus, Streptomyces carbophilus,Streptomyces griseofuscus, Streptomyces thermocoerulescens, Streptomycesnogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes,Streptomyces olivochromogenes, Streptomyces ornatus, Streptomycesgriseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomycespallidus, Streptomyces roseorubens, Streptomyces rutgersensis andStreptomyces steffisburgensis, and more specifically, Streptomycesphaeochromogenes IFO12898, Streptomyces testaceus ATCC21469,Streptomyces achromogenes IFO 12735, Streptomyces griseolus ATCC11796,Streptomyces carbophilus SANK62585, Streptomyces griseofuscus IFO12870t, Streptomyces thermocoerulescens IFO 142731, Streptomycesnogalater IFO 13445, Streptomyces tsusimaensis IFO 13782, Streptomycesglomerochromogenes IFO 13673t, Streptomyces olivochromogenes IFO 12444,Streptomyces ornatus IFO 13069, Streptomyces griseus ATCC 10137,Streptomyces griseus IFO 13849T, Streptomyces lanatus IFO 12787T,Streptomyces misawanensis IFO 13855T, Streptomyces pallidus IFO 13434T,Streptomyces roseorubens IFO 13682T, Streptomyces rutgersensis IFO15875T and Streptomyces steffisburgensis IFO 13446T, and the like; ormicroorganisms belonging to Saccharopolyspora, such as Saccharopolysporataberi, more specifically, Saccharopolyspora taberi JCM 9383t and thelike. The PCR may also utilize an oligonucleotide comprising at leastabout 20 nucleotides of the 5′ terminus of the nucleotide sequenceencoding an amino acid sequence shown in any one of SEQ ID NO: 1, 2, 3,4, 5, 108, 159, 160, 136, 137, 138, 215, 216, 217, 218, 219, 220, 221,222, 223 or 224, with an oligonucleotide comprising a nucleotidesequence comnplimentary to at least about 20 nucleotides adjacent to 3′terminus or downstream of the 3′ terminus of the nucleotide sequenceencoding any one of the amino acid sequences above. The PCR may beconducted under the conditions described below. On the 5′ terminus sideof the primer utilized for the PCR as described above, a restrictionenzyme recognition sequence may be added.

More specifically for example, a DNA comprising a nucleotide sequenceencoding the amino acid sequence shown in SEQ ID NO: 1, a DNA comprisingthe nucleotide sequence shown in SEQ ID NO: 6, or the like can beprepared by conducting PCR by utilizing as the template the chromosomalDNA or chromosomal DNA library prepared from Streptomycesphaeochromogenes IFO 12898 and by utilizing as primers anoligonucleotide comprising the nucleotide sequence shown in SEQ ID NO:51 and an oligonucleotide comprising the nucleotide sequence shown inSEQ ID NO: 52. Alternatively, a DNA comprising the nucleotide sequenceshown in SEQ ID NO: 9 (containing a nucleotide sequence encoding theamino acid sequence shown in SEQ ID NO: 1) can be amplified byconducting PCR by utilizing as primers the oligonucleotide comprisingthe nucleotide sequence shown in SEQ ID NO: 51 and an oligonucleotidecomprising the nucleotide sequence shown in SEQ ID NO: 53.

For example, a DNA comprising a nucleotide sequence encoding the aminoacid sequence shown in SEQ ID NO: 2, a DNA comprising the nucleotidesequence shown in SEQ ID NO: 7, or the like can be prepared byconducting PCR by utilizing as the template the chromosomal DNA orchromosomal DNA library prepared from Saccharopolyspora taberi JCM 9383tand by utilizing as primers an oligonucleotide comprising the nucleotidesequence shown in SEQ ID NO: 61 and an oligonucleotide comprising thenucleotide sequence shown in SEQ ID NO: 62. Alternatively, a DNAcomprising the nucleotide sequence shown in SEQ ID NO: 10 (containing anucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2) can be amplified by conducting PCR by utilizing as primers theoligonucleotide comprising the nucleotide sequence shown in SEQ ID NO:61 and an oligonucleotide comprising the nucleotide sequence shown inSEQ ID NO: 63.

For example, a DNA comprising a nucleotide sequence encoding the aminoacid sequence shown in SEQ ID NO: 108, a DNA comprising the nucleotidesequence shown in SEQ ID NO: 109, or the like can be prepared byconducting PCR by utilizing as the template the chromosomal DNA orchromosomal DNA library prepared from Streptomyces achromogenes IFO12735 and by utilizing as primers an oligonucleotide comprising thenucleotide sequence shown in SEQ ID NO: 119 and an oligonucleotidecomprising the nucleotide sequence shown in SEQ ID NO: 120.Alternatively, a DNA comprising the nucleotide sequence shown in SEQ IDNO: 110 (containing a nucleotide sequence encoding the amino acidsequence shown in SEQ ID NO: 108) can be amplified by conducting PCR byutilizing as primers the oligonucleotide comprising the nucleotidesequence shown in SEQ ID NO: 119 and an oligonucleotide comprising thenucleotide sequence shown in SEQ ID NO: 121.

For example, a DNA comprising the nucleotide sequence shown in SEQ IDNO: 144 (containing a nucleotide sequence encoding the amino acidsequence shown in SEQ ID NO: 159) can be prepared by conducting PCR byutilizing as the template the chromosomal DNA or chromosomal DNA libraryprepared from Streptomyces nogalater IFO 13445 and by utilizing asprimers an oligonucleotide comprising the nucleotide sequence shown inSEQ ID NO: 165 and an oligonucleotide comprising the nucleotide sequenceshown in SEQ ID NO: 166.

For example, a DNA comprising the nucleotide sequence shown in SEQ IDNO: 145 (containing a nucleotide sequence encoding the amino acidsequence shown in SEQ ID NO: 160) can be prepared by conducting PCR byutilizing as the template the chromosomal DNA or chromosomal DNA libraryprepared from Streptomyces tsusimaensis IFO 13782 and by utilizing asprimers an oligonucleotide comprising the nucleotide sequence shown inSEQ ID NO: 171 and an oligonucleotide comprising the nucleotide sequenceshown in SEQ ID NO: 172.

For example, a DNA comprising the nucleotide sequence shown in SEQ IDNO: 146 (containing a nucleotide sequence encoding the amino acidsequence shown in SEQ ID NO: 136) can be prepared by conducting PCR byutilizing as the template the chromosomal DNA or chromosomal DNA libraryprepared from Streptomyces thermocoerulescens IFO14273t and by utilizingas primers an oligonucleotide comprising the nucleotide sequence shownin SEQ ID NO: 177 and an oligonucleotide comprising the nucleotidesequence shown in SEQ ID NO: 178.

For example, a DNA comprising the nucleotide sequence shown in SEQ IDNO: 147 (containing a nucleotide sequence encoding the amino acidsequence shown in SEQ ID NO: 137) can be prepared by conducting PCR byutilizing as the template the chromosomal DNA or chromosomal DNA libraryprepared from Streptomyces glomerochromogenes IFO13673t and by utilizingas primers an oligonucleotide comprising the nucleotide sequence shownin SEQ ID NO: 183 and an oligonucleotide comprising the nucleotidesequence shown in SEQ ID NO: 184.

For example, a DNA comprising the nucleotide sequence shown in SEQ IDNO: 148 (containing a nucleotide sequence encoding the amino acidsequence shown in SEQ ID NO: 138) can be prepared by conducting PCR byutilizing as the template the chromosomal DNA or chromosomal DNA libraryprepared from Streptomyces olivochromogenes IFO 12444 and by utilizingas primers an oligonucleotide comprising the nucleotide sequence shownin SEQ ID NO: 184 and an oligonucleotide comprising the nucleotidesequence shown in SEQ ID NO: 185.

When utilizing as the template the DNA library in which the chromosomalDNA is introduced into the vector, for example, the present DNA (A) canalso be amplified by conducting PCR by utilizing as primers anoligonucleotide comprising a nucleotide sequence selected from anucleotide sequence encoding any one of the amino acid $ sequences shownin SEQ ID NO: 1, 2, 3, 4, 5, 108, 159, 160, 136, 137 or 138 (forexample, an oligonucleotide comprising a nucleotide sequence of at leastabout 20 nucleotides of the 5′ terminus side of the nucleotide sequenceencoding the amino acid sequence shown in SEQ ID NO: 1) and anoligonucleotide of at least about 20 nucleotides comprising a nucleotidesequence complimentary to the nucleotide sequence adjacent to the DNAinsertion site of the vector utilized to construct the library. On sideof the 5′ terminus of the primer utilized for the PCR as describedabove, a restriction enzyme recognition sequence may be added.

As the conditions for the such PCR described above, specifically forexample, there can be mentioned the condition of maintaining 97° C. for2 minutes, then repeating for 10 cycles a cycle that includesmaintaining 97° C. for 15 seconds, followed by 65° C. for 30 seconds,and then 72° C. for 2 minutes; then conducting for 15 cycles a cyclethat includes maintaining 97° C. for 15 seconds, followed by 68° C. for30 seconds, and followed by 72° C. for 2 minutes (adding 20 seconds toevery cycle in turn); and then maintaining 72° C. for 7 minutes. The PCRcan utilize a reaction solution of 50 μl, containing 50 ng ofchromosomal DNA, containing 300 nM of each of the 2 primers in suchpairings described above, containing 5.0 μl of dNTP mixture (a mixtureof 2.0 nM each of the 4 types of dNTPs), containing 5.0 μl of 10× ExpandHF buffer (containing MgCl₂, Roche Molecular Biochemicals Company) andcontaining 0.75 μl of Expand HiFi enzyme mix (Roche MolecularBiochemicals Company).

Alternatively, there can be mentioned the condition of maintaining 97°C. for 2 minutes, then repeating for 30 cycles a cycle that includes 97°C. for 15 seconds, followed by 60° C. for 30 seconds, and followed by72° C. for 90 seconds, and then maintaining the reaction solution at 72°C. for 4 minutes. The PCR can utilize a reaction solution of 50 μlcontaining 250 ng of chromosomal DNA, containing 200 nM of each of the 2primers in such pairings described above, containing 5.0 μl of dNTPmixture (a mixture of 2.5 mM each of the 4 types of dNTPs), 5.0 μl of10× ExTaq buffer (containing MgCl₂, Takara Shuzo Company) and containing0.5 μl of ExTaq Polymerase (Takara Shuzo Company).

Alternatively, for example, oligonucleotides can be designed andprepared for use as primers, based on the nucleotide sequence of aregion to which the sequence identity is particularly high in thenucleotide sequence shown in SEQ ID NO: 6, 7, 8 or 109. The present DNA(A) can also be obtained by conducting PCR by utilizing the obtainedoligonucleotides as primers and a chromosomal DNA or chromosomal DNAlibrary. The chromosomal DNA or chromosomal DNA library can be preparedas described above from microorganisms belonging to Streptomyces, suchas Streptomyces phaeochromogenes, Streptomyces testaceus, Streptomycesachromogenes, Streptomyces griseolus, Streptomyces carbophilus,Streptomyces griseofuscus, Streptomyces thermocoerulescens, Streptomycesnogalater, Streptomyces tsusimaensis, Streptomyces glomerochromogenes,Streptomyces olivochromogenes, Streptomyces ornatus, Streptomycesgriseus, Streptomyces lanatus, Streptomyces misawanensis, Streptomycespallidus, Streptomyces roseorubens, Streptomyces rutgersensis andStreptomyces steffisburgensis, and more specifically, Streptomycesphaeochromogenes IFO12898, Streptomyces testaceus ATCC21469,Streptomyces achromogenes IFO 12735, Streptomyces griseolus ATCC11796,Streptomyces carbophilus SANK62585, Streptomyces griseofuscus IFO12870t, Streptomyces thermocoerulescens IFO 14273t, Streptomycesnogalater IFO 13445, Streptomyces tsusimaensis IFO 13782, Streptomycesglomerochromogenes IFO 13673t, Streptomyces olivochromogenes IFO 12444,Streptomyces ornatus IFO 13069t, Streptomyces griseus ATCC 10137,Streptomyces griseus IFO 13849T, Streptomyces lanatus IFO 12787T,Streptomyces misawanensis IFO 13855T, Streptomyces pallidus IFO 13434T,Streptomyces roseorubens IFO 13682T, Streptomyces rutgersensis IFO15875T and Streptomyces steffisburgensis IFO 13446T, and the like; ormicroorganisms belonging to Saccharopolyspora, such as Saccharopolysporataberi, more specifically, Saccharopolyspora taberi JCM 9383t and thelike. As the “region to which the sequence identity is particularly highin the nucleotide sequence shown in SEQ ID NO: 6, 7, 8 or 109,” forexample, there is mentioned the region corresponding to the region shownwith each of nucleotides 290 to 315, 458 to 485, 496 to 525 or 1046 to1073 in the nucleotide sequence shown in SEQ ID NO: 6. As the primersdesigned on the basis of such regions of the nucleotide sequence, forexample, there can be mentioned a primer comprising the nucleotidesequence shown in any one of SEQ ID NO: 124 to 129.

-   -   SEQ ID NO: 124; based on the nucleotide sequence of the region        corresponding to the region shown with the above nucleotides 290        to 315;    -   SEQ ID NO: 125; based on the nucleotide sequence of the region        corresponding to the region shown with the above nucleotides 458        to 485;    -   SEQ ID NO: 126; based on the nucleotide sequence of the region        corresponding to the region shown with the above nucleotides 458        to 485;    -   SEQ ID NO: 127; based on the nucleotide sequence of the region        corresponding to the region shown with the above nucleotides 496        to 525;    -   SEQ ID NO: 128; based on the nucleotide sequence of the region        corresponding to the region shown with the above nucleotides 496        to 525; and    -   SEQ ID NO: 129; based on the nucleotide sequence of the region        corresponding to the region shown with the above nucleotides        1046 to 1073.

For example, a DNA of approximately 800 bp is amplified by utilizing asprimers the pairing of the oligonucleotide comprising the nucleotidesequence shown in SEQ ID NO: 124 and the oligonucleotide comprising thenucleotide sequence shown in SEQ ID NO: 129. A DNA of approximately 600bp is amplified by utilizing as primers the pairing of theoligonucleotide comprising the nucleotide sequence shown in SEQ ID NO:125 and the oligonucleotide comprising the nucleotide sequence shown inSEQ ID NO: 129. A DNA of approximately 600 bp is amplified by utilizingas primers the pairing of the oligonucleotide comprising the nucleotidesequence shown in SEQ ID NO: 126 and the oligonucleotide comprising thenucleotide sequence shown in SEQ ID NO: 129. A DNA of approximately 580bp is amplified by utilizing as primers the pairing of theoligonucleotide comprising the nucleotide sequence shown in SEQ ID NO:127 and the oligonucleotide comprising the nucleotide sequence shown inSEQ ID NO: 129. Further, a DNA of approximately 580 bp is amplified byutilizing as primers the pairing of the oligonucleotide comprising thenucleotide sequence shown in SEQ ID NO: 128 and the oligonucleotidecomprising the nucleotide sequence shown in SEQ ID NO: 129.

As the conditions for PCR, specifically for example, there is mentionedthe condition of maintaining 95° C. for 1 minute; repeating for 30cycles a cycle that includes maintaining 94° C. for 15 seconds, followedby 60° C. for 30 seconds, and followed by 72° C. for 1 minute; and thenmaintaining 72° C. for 5 minutes. There can be utilized the reactionsolution of 25 μl containing long of chromosomal DNA, containing 200 nMof each of the 2 primers, containing 0.5 μl of dNTP mix (a mixture of 10mM each of the 4 types of dNTPs), containing 5 μl of 5×GC genomic PCRreaction buffer, containing 5 μl of 5M GC-Melt and containing 0.5 μl ofAdvantage-GC genomic polymerase mix (Clontech Company).

By recovering the DNA amplified as described above, a DNA comprising apartial nucleotide sequence of the present DNA (A) can be obtained.Next, based on the nucleotide sequence possessed by the obtained “DNAcomprising a partial nucleotide sequence of the present DNA (A)”, thereis designed and prepared an oligonucleotide comprising a partialnucleotide sequence of at least about 20 nucleotides of said nucleotidesequence or an oligonucleotide comprising a nucleotide sequencecomplimentary to the partial nucleotide sequence of at least about 20nucleotides of said nucleotide sequence. A DNA comprising a partialnucleotide sequence of the present DNA (A) extended downstream of the 3′terminus or upstream of the 5′ terminus of the “DNA comprising a partialnucleotide sequence of the present DNA (A)” obtained as described abovecan be obtained by conducting PCR. The PCR may utilize as primers apairing of an oligonucleotide prepared as described above based on thenucleotide sequence of the “DNA comprising a partial nucleotide sequenceof the present DNA (A)” and an oligonucleotide of at least about 20nucleotides comprising a nucleotide sequence of the region adjacent tothe DNA insertion site of the vector utilized to construct the abovelibrary or an oligonucleotide of at least about 20 bp comprising anucleotide sequence complimentary to such nucleotide sequence thereof.The PCR may, for example, utilize as the template the chromosomal DNAlibrary prepared from the microorganisms which have the ability toconvert compound (II) to compound (III), as described above. Byconnecting such DNA comprising the partial nucleotide sequence of thepresent DNA (A), there can be obtained the present DNA (A). In such aproduction method, there can be utilized a commercially available kit,such as the Universal Genome Walker (Clontech Company). Alternatively,the present DNA (A) can be obtained by conducting PCR by preparingprimers based on the full length nucleotide sequence of the present DNA(A) obtained by connecting the partial nucleotide sequences of thepresent DNA (A) as described above, by utilizing such primers and byutilizing as the template the chromosomal DNA library as describedabove.

For example, a DNA comprising the nucleotide sequence shown innucleotides 316 to 1048 of SEQ ID NO: 139 (a partial nucleotide sequenceof nucleotide sequence encoding the amino acid sequence shown in SEQ IDNO: 159), can be prepared by conducting PCR by utilizing as the templatethe chromosomal DNA or chromosomal DNA library prepared fromStreptomyces nogalater IFO 13445 and by utilizing as primers anoligonucleotide comprising the nucleotide sequence shown in SEQ ID NO:124 and an oligonucleotide comprising the nucleotide sequence shown inSEQ ID NO: 129. A DNA comprising a nucleotide sequence extendeddownstream of the 3′ terminus or upstream of the 5′ terminus thereof isobtained according to the above description based on the nucleotidesequence of the obtained DNA. A DNA comprising the nucleotide sequenceshown in SEQ ID NO: 144 (containing a nucleotide sequence encoding theamino acid sequence shown in SEQ ID NO: 159 and the nucleotide sequenceencoding the amino acid sequence shown in SEQ ID NO: 149) can beobtained by connecting the resulting DNA.

For example, a DNA comprising the nucleotide sequence shown innucleotides 364 to 1096 of SEQ ID NO: 140 (a partial nucleotide sequenceof nucleotide sequence encoding the amino acid sequence shown in SEQ IDNO: 160), can be prepared by conducting PCR by utilizing as the templatethe chromosomal DNA or chromosomal DNA library prepared fromStreptomyces tsusimaensis IFO 13782 and by utilizing as primers anoligonucleotide comprising the nucleotide sequence shown in SEQ ID NO:124 and an oligonucleotide comprising the nucleotide sequence shown inSEQ ID NO: 129. A DNA comprising a nucleotide sequence extendeddownstream of the 3′ terminus or upstream of the 5′ terminus thereof isobtained according to the above description based on the nucleotidesequence of the obtained DNA. A DNA comprising the nucleotide sequenceshown in SEQ ID NO: 145 (containing a nucleotide sequence encoding theamino acid sequence shown in SEQ ID NO: 150 and the nucleotide sequenceencoding the amino acid sequence shown in SEQ ID NO: 160) can beobtained by connecting the resulting DNA.

For example, a DNA comprising the nucleotide sequence shown innucleotides 295 to 1027 of SEQ ID NO: 141 (a partial nucleotide sequenceof nucleotide sequence encoding the amino acid sequence shown in SEQ IDNO: 136), can be prepared by conducting PCR by utilizing as the templatethe chromosomal DNA or chromosomal DNA library prepared fromStreptomyces thermocoerulescens IFO 14273t and by utilizing as primersan oligonucleotide comprising the nucleotide sequence shown in SEQ IDNO: 124 and an oligonucleotide comprising the nucleotide sequence shownin SEQ ID NO: 129. A DNA comprising a nucleotide sequence extendeddownstream of the 3′ terminus or upstream of the 5′ terminus thereof isobtained according to the above description based on the nucleotidesequence of the obtained DNA. A DNA comprising the nucleotide sequenceshown in SEQ ID NO: 146 (containing a nucleotide sequence encoding theamino acid sequence shown in SEQ ID NO: 136 and the nucleotide sequenceencoding the amino acid sequence shown in SEQ ID NO: 151) can beobtained by connecting the resulting DNA.

For example, a DNA comprising the nucleotide sequence shown innucleotides 316 to 1048 of SEQ ID NO: 142 (a partial nucleotide sequenceof nucleotide sequence encoding the amino acid sequence shown in SEQ IDNO: 137), can be prepared by conducting PCR by utilizing as the templatethe chromosomal DNA or chromosomal DNA library prepared fromStreptomyces glomerochromogenes IFO 13673t and by utilizing as primersan oligonucleotide comprising the nucleotide sequence shown in SEQ IDNO: 124 and an oligonucleotide comprising the nucleotide sequence shownin SEQ ID NO: 129. A DNA comprising a nucleotide sequence extendeddownstream of the 3′ terminus or upstream of the 5′ terminus thereof isobtained according to the above description based on the nucleotidesequence of the obtained DNA. A DNA comprising the nucleotide sequenceshown in SEQ ID NO: 147 (containing a nucleotide sequence encoding theamino acid sequence shown in SEQ ID NO: 137 and the nucleotide sequenceencoding the amino acid sequence shown in SEQ ID NO: 152) can beobtained by connecting the resulting DNA.

For example, a DNA comprising the nucleotide sequence shown innucleotides 316 to 1048 of SEQ ID NO: 143 (a partial nucleotide sequenceof nucleotide sequence encoding the amino acid sequence shown in SEQ IDNO: 138), can be prepared by conducting PCR by utilizing as the templatethe chromosomal DNA or chromosomal DNA library prepared fromStreptomyces olivochromogenes IFO 12444 and by utilizing as primers anoligonucleotide comprising the nucleotide sequence shown in SEQ ID NO:124 and an oligonucleotide comprising the nucleotide sequence shown inSEQ ID NO: 129. A DNA comprising a nucleotide sequence extendeddownstream of the 3′ terminus or upstream of the 5′ terminus thereof isobtained according to the above description based on the nucleotidesequence of the obtained DNA. A DNA comprising the nucleotide sequenceshown in SEQ ID NO: 148 (containing a nucleotide sequence encoding theamino acid sequence shown in SEQ ID NO: 138 and the nucleotide sequenceencoding the amino acid sequence shown in SEQ TD NO: 153) can beobtained by connecting the resulting DNA.

For example, a DNA comprising the nucleotide sequence shown innucleotides 289 to 1015 of SEQ ID NO: 232 (a partial nucleotide sequenceof nucleotide sequence encoding the amino acid sequence shown in SEQ IDNO: 222), can be prepared by conducting PCR by utilizing as the templatethe chromosomal DNA or chromosomal DNA library prepared fromStreptomyces roseorubens IFO 13682T and by utilizing as primers anoligonucleotide comprising the nucleotide sequence shown in SEQ ID NO:124 and an oligonucleotide comprising the nucleotide sequence shown inSEQ ID NO: 129. A DNA comprising a nucleotide sequence extendeddownstream of the 3′ terminus or upstream of the 5′ terminus thereof isobtained according to the above description based on the nucleotidesequence of the obtained DNA. A DNA comprising the nucleotide sequenceshown in SEQ ID NO: 242 (containing a nucleotide sequence encoding theamino acid sequence shown in SEQ ID NO: 232 and the nucleotide sequenceencoding the amino acid sequence shown in SEQ ID NO: 252) can beobtained by connecting the resulting DNA.

For example, a DNA comprising the nucleotide sequence shown innucleotides 289 to 1015 of SEQ ID NO: 234 (a partial nucleotide sequenceof nucleotide sequence encoding the amino acid sequence shown in SEQ IDNO: 224), can be prepared by conducting PCR by utilizing as the templatethe chromosomal DNA or chromosomal DNA library prepared fromStreptomyces steffisburgensis IFO 13446T and by utilizing as primers anoligonucleotide comprising the nucleotide sequence shown in SEQ ID NO:124 and an oligonucleotide comprising the nucleotide sequence shown inSEQ ID NO: 129. A DNA comprising a nucleotide sequence extendeddownstream of the 3′ terminus or upstream of the 5′ terminus thereof isobtained according to the above description based on the nucleotidesequence of the obtained DNA. A DNA comprising the nucleotide sequenceshown in SEQ ID NO: 244 (containing a nucleotide sequence encoding theamino acid sequence shown in SEQ ID NO: 234 and the nucleotide sequenceencoding the amino acid sequence shown in SEQ ID NO: 254) can beobtained by connecting the resulting DNA.

The present DNA (A) obtained by utilizing the PCR described above can becloned into a vector by a method according to conventional geneticengineering methods described in “Molecular Cloning: A Laboratory Manual2nd edition” (1989), Cold Spring Harbor Laboratory Press, “CurrentProtocols in Molecular Biology” (1987), John Wiley & Sons, Incorporatedand the like. Specifically for example, cloning can be conducted byutilizing plasmid vectors such as pBluescpriptII of Strategene Companyor a plasmid vector contained in the TA Cloning Kit of InvitrogenCompany.

Further, the present DNA (A) can be prepared, for example, as describedbelow. First a nucleotide sequence is designed. The nucleotide sequenceencodes an amino acid sequence of a protein encoded by the present DNA(A). The nucleotide sequence has a GC content of at most 60% and atleast 40%, preferably at most 55% and at least 45%. The codon usage inthe nucleotide sequence encoding the amino aid sequence of the aboveprotein is within the range of plus or minus 4% of the codon usage ingenes from the species of a host cell to which the present DNA (A) isintroduced. By preparing a DNA having the designed nucleotide sequenceaccording to conventional genetic engineering methods, the present DNA(A) can be obtained.

For example, there can be designed in the way described below, anucleotide sequence encoding an amino acid sequence (SEQ ID NO: 1) ofthe present invention protein (A1) and having a GC content of at most55% and at least 45%, where the codon usage in the nucleotide sequenceencoding the amino acid sequence of the above protein is within therange of plus or minus 4% of the codon usage in genes from soybean.First for example, the codon usage (Table 22 and Table 23) in thenucleotide sequence (SEQ ID NO: 6) encoding the amino acid sequence ofthe present invention protein (A1) which can be obtained fromStreptomyces phaeochromogenes IFO12898 and soybean codon usage (Table 24and Table 25) are compared. Based on the result of the comparison,nucleotide substitutions are added to the nucleotide sequence shown inSEQ ID NO: 6, so that the GC content is at most 55% and at least 45% andthe codon usage is within the range of plus or minus 4% of the soybeancodon usage. As such a nucleotide substitution, there is selected anucleotide substitution which does not result in an amino acidsubstitution. For example, the usage of the CTG codon encoding leucineis 1.22% in soybean genes and 7.09% in the nucleotide sequence shown inSEQ ID NO: 6. As such, for example, each of the CTG codons starting fromnucleotides 106, 163, 181, 226, 289, 292, 544, 1111, and 1210 of thenucleotide sequence shown in SEQ ID NO: 6 is substituted to CTT codons;each of the CTG codons starting from nucleotides 211, 547 and 1084 issubstituted to CTA codons; the CTG codon starting from nucleotide 334 issubstituted to a TTA codon; each of the CTG codons starting fromnucleotides 664, 718, 733, 772, 835, 1120 and 1141 is substituted to aTTG codon; and the CTG codon starting from nucleotide 787 is substitutedto a TTA codon. One sequence of a nucleotide sequence designed in such away is shown in SEQ ID NO: 214, the codon usage in which is shown inTable 26 and Table 27. In the, nucleotide sequence shown it SEQ ID NO:214, for example, the usage of the CTG codon encoding leucine is 1.71%and is within the range of plus or minus 4% of the codon usage (1.22%)for soybean. The DNA comprising the nucleotide sequence shown in SEQ IDNO: 214 can be prepared by introducing nucleotide substitutions to theDNA having the nucleotide sequence shown in SEQ ID NO: 6, according tosite-directed mutagenesis methods described in such as Sambrook, J.,Frisch, E. F., and Maniatis, T.; Molecular Cloning 2nd Edition, ColdSpring Harbor Press. Alternatively, the DNA having the nucleotidesequence shown in SEQ ID NO: 214 can be prepared by a DNA synthesismethod employing the PCR described in Example 46 below.

Similarly, the nucleotide sequence shown in SEQ ID NO: 368 is an exampleof designing a nucleotide sequence encoding the amino acid sequence (SEQID NO: 222) of the present invention protein (A23) and having a CCcontent of at most 55% and at least 45%, where the codon usage in thenucleotide sequence encoding the amino acid sequence of the aboveprotein is within the rage of plus or minus 4% with the codon usage forgenes from soybean. Further, the nucleotide sequence shown in SEQ ID NO:393 is an example of designing a nucleotide sequence encoding the aminoacid sequence (SEQ ID NO: 224) of the present invention protein (A25)and having a GC content of at most 55% and at least 45%, where the codonusage in the nucleotide sequence encoding the amino acid sequence of theabove protein is within the edge of plus or minus 4% with the codonusage for genes from soybean.

The present DNA (A) obtained in such a way can be cloned into a vectoraccording to conventional genetic engineering methods described in suchas Sambrook, J., Frisch, E. F., and Maniatis, T.; “Molecular Cloning 2ndEdition” (1989), Cold Spring Harbor Press; “Current Protocols inMolecular Biology” (1987), John Wiley & Sons, Incorporated, and thelike. As the vector, specifically for example, there can be utilized pUC119 (TaKaRa Shuzo Company), pTVA 118N (Takara Shuzo Company),pBluescript II (Toyobo Company), pCR2.1-TOPO (Invitrogen), pTre99A(Pharmacia Company), pKK331-1A (Pharmacia Company), and the like.

Further, the nucleotide sequence of the present DNA (A) obtained in sucha way can be analyzed by the dideoxy terminator method described in F.Sanger, S Nicklen, A. R. Coulson, Proceeding of National Academy ofScience U.S.A. (1977) 74:5463-5467.

The ability to metabolize the PPO inhibitory-type herbicidal compound offormula (I) of the present protein (A), which is encoded by the presentDNA (A) obtained in such a way described above, can be confirmed withthe ability of converting compound (II) to compound (III) as a marker inthe way described below. First, as described below, said DNA is insertedinto a vector so that it is connected downstream of a promoter which canfunction in the host cell and that is introduced into a host cell toobtain a transformant. Next, the culture of the transformant or theextract obtained from disrupting the culture is reacted with compound(II) in the presence of an electron transport system containing anelectron donor, such as coenzyme NADPH. The reaction products resultingtherefrom are analyzed to detect compound (III). In such a way, therecan be detected a transformant having the ability of metabolizingcompound (II) and producing compound (III), and be determined that sucha transformant bears the present DNA (A) encoding the protein havingsuch ability. More specifically for example, there is prepared 30 μl ofa reaction solution consisting of a 0.1M potassium phosphate buffer (pH7.0) comprising the culture or extract of the above transformant, anelectron donor such an β-NADPH at a final concentration of about 2 mM,ferredoxin derived from spinach at a final concentration of about 2mg/ml, ferredoxin reductase at a final concentration of about 0.1 U/mland 3 ppm of compound (II) labeled with a radioisotope. The reactionsolution is incubated at about 30° C. to 40° C. for 10 minutes to 1hour. After such incubation, 3 μl of 2N HCl and 90 μl of ethyl acetateare added, stirred and centrifuged at 8,000 g to recover thesupernatant. After drying the supernatant in vacuo, the residue isdissolved in ethyl acetate and the obtained solution is developed on asilica gel TLC plate. The TLC plate is analyzed by analyzed autography.By identifying the spots corresponding to compound (III) labeled with aradioisotope, there can be confirmed the ability to convert compound(II) to compound (III).

A DNA encoding a protein having the ability to convert compound (II) tocompound (III) or a microorganism having such a DNA may be furthersearched by conducting the hybridizations or PCR as described above,utilizing the present invention DNA (A) or the polynucleotide comprisinga partial nucleotide sequence of said DNA or a nucleotide sequencecomplimentary to the partial nucleotide sequence.

Specifically for example, hybridization as described above is conductedand the DNA to which a probe is hybridized is identified. Thehybridization is conducted with the use of the present invention DNA (A)or a polynucleotide comprising a partial nucleotide sequence of thepresent invention DNA (A) of a nucleotide sequence complimentary to thepartial nucleotide sequence as a probe, and genomic DNA derived from anatural microorganism, for example, microorganisms belonging tostreptomyces such as Streptomyces phaeochromogenes, Streptomycestestaceus, Streptomyces achromogenes, Streptomyces griseolus,Streptomyces carbophilus, Streptomyces griseofuscus, Streptomycesthermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis,Streptomyces glomerochromogenes, Streptomyces olivochromogenes,Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus,Streptomyces misawanensis, Streptomyces pallidus. Streptomycesroseorubens, Streptomyces rutgersensis and Streptomycessteffisburgensis; microorganisms belonging to Saccharopolyspora such asSaccharopolyspora taberi; and the like. As specific examples of DNAwhich can be utilized as the probe, there can be mentioned a DNAcomprising the full length of the nucleotide sequence shown in any oneof SEQ ID NO; 6, 7, 8, 109, 139, 140, 141, 142, 143, 225, 226, 227, 228,229, 230, 231, 232, 273 or 234; a DNA comprising a nucleotide sequenceshown in nucleotides 57 to 730 of the nucleotide sequence shown in SEQID NO: 6; a DNA comprising a nucleotide sequence show in nucleotides 21to 691 of the nucleotide sequence shown in SEQ ID NO: 8; and the like.

Alternatively, PCR can be conducted as described above and the amplifiedDNA can be detected. The PCR utilizes a polynucleotide comprising apartial nucleotide sequence if the present invention DNA (A) or anucleotide sequence complimentary to the partial nucleotide sequence.The PCR utilizes as the template genomic DNA derived from a naturalmicroorganism, for example, microorganisms belonging to streptomycessuch as Streptomyces phaeochromogenes, Streptomyces testaceus,Streptomyces achromogenes, Streptomyces griseolus, Streptomycescarbophilus, Streptomyces griseofuscus, Streptomyces thermocoerulescens,Streptomyces nogalater, Streptomyces tsusimaensis, Streptomycesglomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus,Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis,Streptomyces pallidus. Streptomyces roseorubens, Streptomycesrutgersensis and Streptomyces steffisburgensis; microorganisms belongingto Saccharopolyspora such as Saccharopolyspora taberi; and the like. Asthe primers, there can be mentioned primers which were designed, basedon the nucleotide sequence of the “region to which the sequence identityis particularly high in the nucleotide sequence shown in SEQ ID NO: 6,7, 8 or 109” as described above. As more specific examples of theprimers, there is mentioned pairings of a primer comprising a nucleotidesequence shown in any one of SEQ ID NO: 124 to 128 and a primercomprising a nucleotide sequence shown in SEQ ID NO: 129.

The DNA detected in such a way is recovered. When the recovered DNA doesnot contain the full length nucleotide sequence of the present DNA (A),such DNA is utilized and made into a DNA corresponding to the fulllength nucleotide sequence in a way described above. The obtained DNA isintroduced into a host cell to produce a transformant. The ability toconvert compound (II) to compound (III) of the protein encoded by theDNA introduced into the transformant can be evaluated by utilizing theculture of the obtained transformant and measuring the ability toconvert compound (II) to compound (III) in a way described above.

To express the present DNA (A) in a host cell, the present DNA (A) isintroduced into the host cell in a position enabling its expression insaid cell. By introducing the present DNA (A) into a “position enablingits expression”, it means that the present DNA (A) is introduced into ahoist cell so that it is placed in a position adjacent to a nucleotidesequence directed in transcription and translation from the nucleotidesequence thereof (that is, for example, a nucleotide sequence promotingthe production of the present protein (A) and an RNA encoding thepresent protein (A)).

To introduce the present DNA (A) into the host cell so that it is placedin a position enabling its expression, for example, a DNA in which thepresent DNA (A) and a promoter functional in the host cell are operablylinked is introduced into the host cell. The term “operably linked” heremeans that a condition in which the present DNA (A) is linked to apromoter so that it is expressed under the control of the promoter, whenthe DNA is introduced into a host cell.

When the host coli is a microorganism cell, as a functional promoter,for example, there can be mentioned the lactose operon promoter of E.coli, tryptophan operon promoter of E. coli, T7 phage promoter orartificial promoters functional in E. coli such as tac promoter or trcpromoter and the like. Further, there may be utilized the promoteroriginally present upstream of the present DNA (A) in the chromosome ofthe microorganism belonging to Streptomyces or Saccharopolyspora.

When the host cell is a plant cell, as a functional promoter, forexample, there is mentioned T-DNA derived constitutive promoters such asnopaline synthase gene promoter and octopine synthase gene promoter;plant virus-derived promoters such as cauliflower mosaic virus derived19S and 35S promoters; inducible promoters such as phenylalanineammonia-lyase gene promoter, chalcone synthase gene promoter andpathogenesis-related protein gene promoter; the plant promoter describedin Japanese Unexamined Patent Publication No. 2000-166577. Further, aterminator functional in a plant cell may be connected to the DNA inwhich the promoter functional in a plant cell and the present DNA (A)are operably linked. In this case, it is generally preferred that theterminator is connected downstream from the present DNA (A). As thefunctional terminator, for example, there is mentioned T-DNA derivedconstitutive terminators such as nopaline synthase gene (NOS)terminator; plant virus derived terminators such as terminators ofallium virus GV1 or GV2; the plant terminator described in JapaneseUnexamined Patent Publication No. 2000-166577; and the like.

When introducing the present DNA (A) so that the DNA is placed in aposition enabling its expression, for example, there can be utilized aDNA having a nucleotide sequence encoding a transit signal to anintracellular organelle, linked upstream of the present DNA (A), so thatthe reading frames are in frame. By being linked “so that the readingframes are in frame” it means that reading frame of the sequence of thetransit signal to an intracellular organelle and the reading frame ofthe present DNA (A) are connected to form one continuous reading frame.As a transit signal sequence providing the transition and localizationof a protein in an intracellular organelle in a plant cell, for example,there can be mentioned a transit signal derived from a cytoplasmicprecursor of a protein localizing in the chloroplast of a plant asdescribed in U.S. Pat. No. 5,717,084, the chimeric sequences formed fromthe variety of the transit signal sequences described in U.S. Pat. No.RE36449. More specifically, there is mentioned the chloroplast transitpeptide derived from the small subunit of ribulose-1,5-bisphosphatecarboxylase of soybean, which is obtainable according to the methoddescribed in Example 15 below.

Typically, the present DNA (A), the present DNA (A) to which a DNAhaving a nucleotide sequence encoding a transit signal to anintracellular organelle is connected as described above, or a DNA inwhich such DNA is operably linked to a promoter functional in the hostcell, can each be inserted into a vector usable in a host cell and thisis introduced into the host cell. When utilizing a vector alreadypossessing a promoter functional in the host cell, the present DNA (A)may be inserted downstream of a promoter present in the vector so thatsaid promoter and the present DNA (A) can be operably linked.

As the vector, specifically when utilizing E. coli as the host cell, forexample, there can be mentioned pUC 119 (TaKaRa Shuzo Company), pTVA118N (Takara Shuzo Company), pBluescript II (Strategene Company),pCR2.1-TOPO (Invitrogen), pTrc99A (Amersham Pharmacia Biotech Company),pKK331-1A (Amercham Pharmacia Biotech Company), pET11d (Novagen) and thelike. By utilizing a vector containing a selective marker (for example,genes conferring resistance to an antibiotic such as a kanamycinresistance gene, neomycin resistance gene, and the like), it isconvenient in that the transformant to which the present DNA isintroduced can be selected with the phenotype of the selective marker asan indicator.

As the method of introducing the present DNA (A) or a vector containingthe present DNA (A) into a host cell, there can be mentioned the methoddescribed in Shin Seikagaku Zikken Kouza (Nippon-Seikagaku-Kai eds.,Tokyo Kagaku Dozin), Vol. 17, Biseibutu-Zikken-Hou when the host cell isa microorganism, for example, E. coli, Bacillus subtilis, Bacillusbrevis, Pseudomonas sp., Zymomonas sp, lactic acid bacteria, acetic acidbacteria, Staphylococcus sp., Streptomyces sp., Saccharopolyspora sp.,or yeast such as Saccharomyces cerevisiae, Schizosaccaromyces ponmbe,fungus such as Aspergillus, and the like. Alternatively, for example,there may be utilized the calcium chloride method described in Sambrook,J., Frisch, E. F., and Maniatis, T.; “Molecular Cloning 2nd edition”,Cold Spring Harbor Press (Molecular Biology, John Wiley & Sons, N.Y.(1989) or in “Current Protocols in Molecular Biology” (1987), John Wiley& Sons, Incorporated or the electroporation method described in “Methodsin Electroporation: Gene Pulser/E. coli Pulser System”. Bio-RadLaboratories (1993).

The transformant to which the present DNA (A) or the vector containingthe present DNA (A) has been introduced, for example, can be selected byselecting for the phenotype of the selective marker contained in thevector to which the present DNA (A) has been inserted as described aboveas an indicator. Further, whether the transformant contains the presentDNA (A) or a vector containing the present DNA (A) can be confirmed bypreparing the DNA from the transformant and then conducting with theprepared DNA genetic engineering analysis methods described in, forexample, “Molecular Cloning 2nd edition”, Cold Spring Harbor Press(Molecular Biology, John Wiley & Sons, N.Y. (1989) (such as confirmingrestriction enzyme sites, DNA sequencing, southern hybridizations, PCRand the like).

When the host cell is a plant cell, plant types can be mentioned, forexample, dicotyledones such as tobacco, cotton, rapeseed, sugar beet,Arabidopsis, canola, flax, sunflower, potato, alfalfa, lettuce, banana,soybean, pea, legume, pine, poplar, apple, grape, orange, lemon, othercitrus fruits, almond, walnut other nuts; monocotyledones such as corn,rice, wheat, barley, rye, oat, sorghum, sugar cane and lawn; and thelike. As the cell to which the present DNA (A) is introduced there canbe utilized plant tissue, plant body, cultured cells, seeds and thelike.

As methods of introducing the present DNA (A) or the vector containingthe present DNA (A) into a host cell, there is mentioned methods such asinfection with Agrobacterium (Japanese Examined Patent PublicationNo.2-58917 and Japanese Unexamined Patent Publication No. 60-70080),electroporation into protoplasts (Japanese Unexamined Patent PublicationNo. 60251887 and Japanese Unexamined Patent Publication No. 548575) orparticle gun method (Japanese Unexamined Patent Publication No. 5-508316and Japanese Unexamined Patent Publication No. 63-258525).

In such cases, for example, the transformant to which the present DNAhas been introduced can be selected with the phenotype of a selectivemarker as an indicator, by introducing into the plant cell at the sametime with the vector containing the present DNA (A), a selective makerselected from the hygromycin phosphotransferase gene, neomycinphosphotransferase gene and chloramphenicol acetyltransferase gene. Theselective marker gene and the present DNA (A) may be inserted into thesame vector and introduced. A vector comprising the selective markergene and a vector comprising the present DNA (A) may also be introducedat the same time. A transformant to which the present DNA (A) has beenintroduced may also be selected by culturing with a medium containingthe PPO inhibitory-type herbicidal compound of formula (I) and byisolating a clone multipliable therein. Whether the transformantcontains the present DNA (A) can be confirmed by preparing the DNA fromthe transformant and then conducting with the prepared DNA geneticengineering analysis methods described in, for example, “MolecularCloning 2nd edition”, Cold Spring Harbor Press (Molecular Biology, JohnWiley & Sons, N.Y. (1989) (such as confirming restriction enzyme sites,DNA sequencing, southern hybridizations, PCR and the like). The presentDNA (A) introduced in the plant cell may be maintained at locations inthe cell other than the DNA contained in the nucleus, by being insertedinto the DNA contained in intracellular organelles such as thechloroplast.

From the transformed plant cell obtained in such a way, a transgenicplant to which the present DNA (A) has been introduced can be obtained,by regenerating a plant body by the plant cell culturing methoddescribed in Shokubutu-Idenshi-Sosa-Manual:Transgenic-Shokubutu-No-Tukurikata (Uchimiya, Kodansha-Scientific,1990), pp. 27-55. Further, a targeted plant type to which the presentDNA (A) has been introduced can be produced by mating the targeted typeof plant with the transgenic plant to which the present DNA (A) has beenintroduced, so that the present DNA (A) is introduced into a chromosomeof the targeted type of plant.

Specifically, for example, rice or Arabidopsis having introduced thereinthe present DNA (A) and expressing the present protein (A) can beobtained by the method described in Model-Shokubutu-No-Jikken-Protocol.Ine, Shiroinunazuna-Hen (Supervisors: Koh SHIMAMOTO and Kiyotaka OKADA,Shujun-sha, 1996), Fourth chapter. Further, there can be obtained asoybean having introduced therein the present DNA (A) and expressing thepresent protein (A) by an introduction into a soybean somatic embryowith a particle gun according to the method described in JapaneseUnexamined Patent Publication No. 3-291501. Likewise, a maize havingintroduced therein the present DNA (A) and expressing the presentprotein (A) can be obtained by an introduction into maize somatic embryowith a particle gun according to the method described by Fromm, M. E.,et al., Bio/Technology, 8; p 838 (1990). Wheat having introduced thereinthe present DNA (A) and expressing the present protein (A) can beobtained by introducing the gene into sterile-cultured wheat immaturescutellum with a particle gun according to a conventional methoddescribed by TAKUMI et al., Journal of Breeding Society (1995), 44:Extra Vol. 1, p 57. Likewise, barley having introduced therein thepresent DNA (A) and expressing the present protein (A) can be obtainedby an introduction into sterile cultured barley immature scutellum witha particle gun according to a conventional method described by HAGIO, etal., Journal of Breeding Society (1995), 44; Extra Vol. 1, p 67.

The transformant having introduced therein the present DNA (A) andexpressing the present protein (A) can reduce the plant damage bycompound (I), by converting said herbicidal compound into a compound oflower herbicidal activity within its cells. Specifically, for example,by spreading the microorganism expressing the present protein (A) to thecultivation area of the desired cultivated plant before sowing seeds ofthe desired plant, the herbicidal compound remaining in the soil can bemetabolized and the damage to the desired plant can be reduced. Further,by getting the desired variety of plant to express the present protein(A), the ability to metabolize the PPO inhibitory-type herbicidalcompound of formula (I) to a compound of lower activity is conferred tosaid plant. As a result, the plant damage from the herbicidal compoundin the plant is reduced and resistance to said compound is conferred.

The present protein (A) can be prepared, for example, by culturing acell comprising the present DNA (A). As such a cell, there is mentioneda microorganism expressing the present DNA (A) and having the ability toproduce the present protein (A), such as a microorganism strain isolatedfrom nature comprising the present DNA (A), mutant strains derived fromthe natural strain by treatment with agents or ultraviolet rays or thelike. More specifically for example, there is mentioned microorganismsbelonging to Streptomyces, such as Streptomyces phaeochromogenesIFO12898, Streptomyces testaceus ATCC21469, Streptomyces achromogenesIFO 12735, Streptomyces griseolus ATCC11796, Streptomyces carbophilusSANK62585, Streptomyces griseofuscus IFO 12870t, Streptomycesthermocoerulescens IFO 14273t, Streptomyces nogalater IFO 13445,Streptomyces tsusimaensis IFO 13782, Streptomyces glomerochromogenes IFO13673t, Streptomyces olivochromogenes IFO 12444, Streptomyces ornatusIFO 13069t, Streptomyces griseus ATCC 10137, Streptomyces griseus IFO13849T, Streptomyces lanatus IFO 12787T, Streptomyces misawanensis IFO13855T, Streptomyces pallidus IFO 13434T, Streptomyces roseorubens IFO13682T, Streptomyces rutgersensis IFO 15875T and Streptomycessteffisburgensis IDO 13446T, and the like; or microorganisms belongingto Saccharopolyspora, such as Saccharopolyspora taberi JCM 9383t and thelike. Further, there can be mentioned a transformant in which thepresent DNA (A) or a vector containing the present DNA (A) has beenintroduced. Specifically for example, there is mentioned a transformantin which the present DNA (A) operably linked to a tac promoter, trcpromoter, lac promoter or t7 phage promoter is introduced into E. coli.As more specific examples, there is mentioned E. coli JM109/pKSN657, E.coli JM109/pKSN657F, E. coli JM109/pKSN923, E. coli JM109/pKSN923F, E.coli JM109/pKSN11796, E. coli JM109/pKSN11796F, E. coli JM109/pKSN671,E. coli JM109/pKSN671F, E. coli JM109/pKSNSCA, E. coli JM109/pKSN646, E.coli JM109/pKSN646F, E. coli JM109/pKSN849AF, E. coli JM109/pKSN1618F,E. coli JM109/pKSN474F, E. coli JM109/pKSN1491AF, E. coliJM109/pKSN1555AF, E. coli JM109/pKSN1584F, E. coli JM109/pKSN1609F andthe like, described in the examples described below.

As a medium for culturing the above microorganisms comprising thepresent DNA (A), there can be utilized any of those employed usually forculturing a microorganism which contains carbon sources and nitrogensources, organic and inorganic salts as appropriate. A compound which isa precursor to heme, such as aminolevulinic acid, may be added.

As the carbon source, for example, there is mentioned saccharides suchas glucose, fructose, sucrose and dextrin; sugar alcohols such asglycerol and sorbitol; and organic acids such as fumaric acid, citricacid and pyruvic acid; and the like. The amount of carbon sources listedabove to be added to a medium is usually about 0.1% (w/v) to about 10%(w/v) based on a total amount of the medium.

As the nitrogen source, for example, there is mentioned ammonium saltsof inorganic acids such as ammonium chloride, ammonium sulfate andammonium phosphate; ammonium salts of organic acids such as ammoniumfumarate and ammonium citrate; organic nitrogen sources, such as meatextract, yeast extract, malt extract, soybean powder, corn steep liquor,cotton seed powder, dried yeast, casein hydrolysate; as well as aminoacids. Among those listed above, ammonium salts of organic acids,organic nitrogen sources and amino acids may mostly be employed also ascarbon sources. The amount of nitrogen sources to be added is usuallyabout 0.1% (w/v) to about 10% (w/v) based on the total amount of themedium.

As the inorganic salt, for example, there is mentioned phosphates suchas potassium phosphate, dipotassium phosphate, sodium phosphate,disodium phosphate; chlorides such as potassium chloride, sodiumchloride, cobalt chloride hexahydrate; sulfates such as magnesiumsulfate, ferrous sulfate heptahydrate, zinc sulfate heptahydrate,manganese sulfate trihydrate; and the like. The amount to be added isusually about 0.0001% (w/v) to about 1% (w/v) based on a total amount ofthe medium.

In case of culturing a transformant retaining the present DNA (A)connected downstream of a 17 phage promoter and a DNA in which thenucleotide sequence encoding T7 RNA polymerase (λDE3 lysogen) isconnected downstream of a lac UV5 promoter, typically, a small amountof, for example, isopropyl-β-D-thiogalactoside (hereinafter referred toas “IPTG”) may be added as an inducer for inducing the production of thepresent protein (A). IPTG can also be added to the medium in case ofculturing a transformant having introduced therein a DNA in which thepresent DNA (A) is operably linked to a type of promoter which isinduced by lactose, such as tac promoter, trc promoter and lac promoter.

A microorganism comprising the present DNA (A) can be cultivated inaccordance with a method employed usually to culture a microorganism,including a liquid phase cultivation such as a rotatory shakingcultivation, a reciprocal shaking cultivation, a jar fermentation (JarFermenter cultivation) and a tank cultivation; or a solid phasecultivation. When ajar fermenter is employed, aseptic air should beintroduced into the Jar Fermenter usually at an aeration rate of about0.1 to about 2 times culture fluid volume per minute. The temperature atwhich the cultivation is performed may vary within a range allowing amicroorganism to be grown, and usually ranges from about 15° C. to about40° C., and the pH of the medium ranges from about 6 to about 8. Thecultivation time may vary depending on the cultivation conditions, andis usually about 1 day to about 10 days.

The present protein (A) produced by a microorganism comprising thepresent DNA (A), for example, can be utilized in various forms in thetreatment of the PPO inhibitory-type herbicidal compound of formula (I),such as a culture of a microorganism producing the present protein (A),a cell of a microorganism producing the present protein (A), a materialobtained by treating such a cell, a cell-free extract of amicroorganism, a crudely purified protein, a purified protein and thelike. A material obtained by treating a cell described above includesfor example a lyophilized cell, an acetone-dried cell, a ground cell, anautolysate of a cell, an ultrasonically treated cell, an alkali-treatedcell, an organic solvent-treated cell and the like. Alternatively, thepresent protein (A) in any of the various forms described above may beimmobilized in accordance with known methods such as a support bindingmethod employing an adsorption onto an inorganic carrier such as asilica gel or a ceramic material, a polysaccharide derivative such as aDEAE-cellulose, a synthesized polymer such as Amberite IRA-935 (TradeName, manufactured by Rohm and Haas) and the like, and an inclusionmethod employing an inclusion into a network matrix of a polymer such asa polyacrylamide, a sulfur-containing polysaccharide gel (e.g.carrageenan gel), an alginic acid gel, an agar gel and the like, andthen used in the treatment of the herbicidal compound described above.

As methods of purifying the present protein (A) from a culture of amicroorganism comprising the present DNA (A), there can be employedconventional methods utilized in a purification of protein. For example,there can be mentioned the 4 following method.

First, cells are harvested from a culture of a microorganism bycentrifugation or the like, and then disrupted physically by anultrasonic treatment, a DYNOMILL treatment, a FRENCH PRESS treatment andthe like, or disrupted chemically by utilizing a surfactant or acell-lyzing enzyme such as lysozyme. From the resultant lysate thusobtained, insoluble materials are removed by centrifugation, membranefiltration or the like to prepare a cell-free extract, which is thenfractionated by any appropriate means for separation and purification,such as a cation exchange chromatography, an anion exchangechromatography, a hydrophobic chromatography, a gel filtrationchromatography and the like, whereby purifying the present protein (A).Supporting materials employed in such chromatography include for examplea resin support such as cellulose, dextran and agarose connected with acarboxymethyl (CM) group, a diethylaminoethyl (DEAE) group, a phenylgroup or a butyl group. A commercially available column already packedwith any support such as Q-Sepharose FF, Phenyl-Sepharose EP, PD-10 andHiLoad 26/10 Q Sepharose BP (Trade Name, from Amersham PharmaciaBiotech), TSK-gel G3000SW (Trade Name, TOSOH CORPORATION) may also beemployed.

One example of purifying the present protein (A) is given.

Cells of a microorganism producing the present protein (A) are harvestedby centrifugation, and then suspended in a buffer such as 0.1M potassiumphosphate (p[7.0). The suspension is treated ultrasonically to disruptthe cells, and the resultant lysate thus obtained is centrifuged atabout 40,000 g for about 30 minutes to obtain a supernatant, which isthen centrifuged at 150,000 g for about 1 hour to recover thesupernatant (the cell-free extract). The obtained cell-free extract issubjected to ammonium sulfate fractionation to obtain the fraction thatis soluble in the presence of 45%-saturated ammonium sulfate andprecipitates at 55%-saturated ammonium sulfate. After the solvent of thefraction is exchanged with a buffer containing no ammonium sulfate, suchas 1M potassium phosphate, utilizing a PD10 column (Amersham PharmaciaBiotech Company), the resulting lion is loaded, for example, onto aHiLoad 26/10 Q Sepharose HP column (Amersham Pharmacia Biotech Company).The column is eluted with 20 mM bistrispropane with a linear gradient ofNaCl to obtain a series of fractions of eluate. The fractions showingactivity in converting compound (II) to compound (III) in the presenceof an electron transport system containing an electron donor, such ascoenzyme NADPK are recovered. Next, after exchanging the buffer in thefactions by utilizing for example the PD10 column (Amersham PharmaciaBiotech Company), the recovered fractions are loaded onto a Bio-ScaleCeramic, for example, Hydroxyapatite, Type I column CHT10-I (BioRadCompany). After washing the column with Buffer A (2 mM potassiumphosphate buffer containing 1.5 mM of CaCl₂; pH7.0), the column iseluted with Buffer A with a linear gradient of Buffer B (100 mMpotassium phosphate buffer containing 0.03 mM CaCl₂) to obtain a seriesof fractions of eluate. The fractions showing activity in convertingcompound (II) to compound (III) in the presence of an electron transportsystem containing an electron donor, such as coenzyme NADPH, arerecovered. After exchanging the buffer in the fractions by utilizing forexample the PD10 column (Amersham Pharmacia Biotech Company), therecovered fractions are concentrated by for example ultrafiltration(microcon filter unit microcon-30; Millipore Company). The resultingfraction is injected for example into a HiLoad 16/60 Superdex column 75pg column (Amersham Pharmacia Biotech Company) and eluted with a 0.05Mpotassium phosphate buffer containing 0.15M NaCl (pH7.0) to obtain aseries of fractions of eluate. The fractions showing activity inconverting compound (II) to compound (III) in the presence of anelectron transport system containing an electron donor, such as coenzymeNADPH, are recovered. The present protein (A) can be purified by aseparation with an SDS-PAGE as needed.

By purifying the present invention protein (A) in the way describedabove, followed by utilizing the obtained present invention protein (A)as an immune antigen, there can be produced an antibody recognizing thepresent invention protein (A) (hereinafter sometimes referred to as the“present invention antibody (A)”).

Specifically, for example, an animal is immunized with the presentprotein (A) purified in the way described above, as an antigen. Forexample, to immunize an animal such as a mouse, hamster, guinea pig,chicken, rat, rabbit, dog and the like, the antigen is administered atleast once, utilizing a conventional method of immunization describedin, for example, W. H. Newsome, J. Assoc. Off. Anal. Chem. 70(6)1025-1027 (1987). As the schedule of administration, for example, thereis mentioned an administration of 2 or 3 times at 7- to 30-dayintervals, preferably, 12- to 16-day intervals. The dose thereof is, forexample, from about 0.05 mg to 2 mg of the antigen for eachadministration. The administration route may be selected fromsubcutaneous administration, intracutaneous administration,intraperitoneal administration, intravenous administration, andintramuscular administration and an injection given intravenously,intraabdominally or subcutaneously is a typical administration form. Theantigen is typically used after being dissolved in a suitable buffer,for example, sodium phosphate buffer or physiological saline containingat least one type of ordinarily used adjuvant such as complete Freund'sadjuvant (a mixture of Aracel A, Bayol F and dead tubercule bacillus),RAS [MPL (monophosphoryl lipid A)+TDM (synthetic trehalosedicorynomycolate)+CWS (cell wall skeleton) adjuvant system] or aluminumhydroxide. However, depending on the administration route or conditions,the adjuvants described above may not be used. The “adjuvant” is asubstance which upon administration with the antigen, enhances a immunereaction unspecifically against the antigen. After nurturing the animaladministered with the antigen for 0.5 to 4 months, a small amount ofblood is sampled from e.g. an ear vein of the animal and measured forantibody titer When the antibody titer is increasing, then the antigenis further administered for an appropriate number of times, depending oncases. For example, the antigen may be administered for one more time ata dose of about 100 μg to 100 μg. One or two months after the lastadministration, blood is collected in a usual manner from the immunizedanimal. By having the blood fractionated by conventional techniques suchas precipitation by centrifugation or with ammonium sulfate or withpolyethylene glycol, chromatography such as gel filtrationchromatography, ion-exchange chromatography and affinity chromatography,and the like, the present invention antibody (A) may be obtained as apolyclonal antiserum. Further, the antiserum may be incubated e.g. at56° C. for 30 minutes to inactivate the complement system.

Alternatively, a polypeptide comprising a partial amino acid sequence ofthe present invention protein (A) is synthesized chemically andadministered as an immune antigen to an animal, whereby producing thepresent invention antibody (A). As the amino acid sequence of apolypeptide employed as an immune antigen, an amino acid sequence whichhas as a low homology as possible with the amino acid sequences of otherproteins is selected from amino acid sequences of the present inventionprotein (A). A polypeptide having a length of 10 amino acids to 15 aminoacids consisting of the selected amino acid sequence is synthesizedchemically by a conventional method and crosslinked for example with acarrier protein such as Limulus plyhemus hemocyanin using MBS and thelike and then used to immunize an animal such as a rabbit as describedabove.

The resultant present invention antibody (A) is then brought intocontact with a test sample, and then a complex of the protein in thetest sample with the antibody described above is detected by aconventional immunological method, whereby detecting the presentinvention protein (A) or a polypeptide comprising a partial amino acidthereof in the test sample. Specifically, for example, it is possible toevaluate the presence of the present invention protein (A) or toquantify the present invention protein (A) in the examined test sampleby a western blot analysis utilizing the present invention antibody (A)as shown in Examples 45 or 73 described below.

Further, for example, a cell expressing the present protein (A) can bedetected, by contacting the present invention antibody (A) with a testcell or a test sample prepared from the test cell followed by detectinga complex of the above antibody and the protein in the test cell or atest sample prepared from the test cell, according to conventionalimmunology methods. By detecting the cell expressing the presentinvention protein (A) in such a way, it is also possible to select froma variety of cells, a cell expressing the present invention protein (A).It is also possible to clone or select a clone containing the presentinvention protein (A) with the use of the present invention antibody(A). For example, a genomic library can be produced by extractinggenomic DNA from a cell that expresses the present invention protein (A)followed by inserting the genomic DNA into an expression vector. Thegenomic library is introduced into a cell. From the obtained cell group,a cell expressing the present invention protein (A) is selected with theuse of the present invention antibody (A) in the way described above.

A kit comprising the present invention antibody (A) can be utilized todetect the present invention protein (A) as described above or toanalyze, detect or search for a cell expressing the present inventionprotein (A). The kit of the present invention may contain the reagentsnecessary for the above analysis methods, other than the presentinvention antibody (A), and may have such a reagent used together withthe present invention antibody (A).

By reacting a PPO inhibitory-type herbicidal compound of formula (I) inthe presence of an electron transport system containing an electrondonor, such as coenzyme NADPH, with the present protein (A), the abovecompound is metabolized and is converted into a compound of lowerherbicidal activity. Specifically for example, by reacting compound (II)in the presence of an electron transport system containing an electrondonor, such as coenzyme NADPH, with the present protein (A), compound(II) is converted to compound (III), which shows substantially noherbicidal activity. An example of protein (A) in such cases is thepresent invention protein (A). One variation of the present protein (A)may be utilized and multiple variations may be utilized together.

The compound of formula (I) is a compound having a uracil structure. Asspecific examples, there can be mentioned compound (II) or a compound ofany one of formulas (IV) to (IX) (hereinafter, referred respectively toas compound (IV) to compound (IX)). It is possible to synthesizecompound (II) and compound (IX) according to the method described inJapanese Unexamined Patent Publication No. 2000-319264, compound (IV)and compound (V) according to the method described in U.S. Pat. No.5,183,492, compound (VI) according to the method described in U.S. Pat.No. 5,674,810, compound (VI) according to the method described inJapanese Unexamined Patent Publication No, 3-204865, and compound (VII)according to the method described in Japanese Unexamined PatentPublication No. 6-321941.

Further, as specific examples of the compound of formula (I), there canbe mentioned a compound of any one of formulas (X) to (XVII)(hereinafter, respectively referred to as compound (X) to compound(XVII)).

Compounds which can be a substrate of the metabolizing reaction by thepresent protein (A) can be selected by having the compound present in areaction in which compound (II) labeled with a radioisotope is reactedwith the present protein (A), in the presence of an electron transportsystem containing an electron donor, such as coenzyme NADPH, anddetecting as a marker the competitive inhibition of the conversionreaction by the present protein (A) of the labeled compound (U) to thelabeled compound (III). When assaying for the presence of thecompetitive inhibition from a test compound, the test compound istypically added to amount to a molar concentration of from 1 to 100times of the labeled compound (II).

The reaction in which compound (I) is reacted with the present protein(A) can be conducted, for example, in an aqueous buffer containing saltsof inorganic acids such as an alkaline metal phosphate such as sodiumphosphate and potassium phosphate; or salts of organic acids such as analkaline metal acetate such as sodium acetate and potassium acetate; orthe like. The concentration of the compound of formula (I) in ametabolizing reaction solution is typically at most about 30% (w/v) andpreferably about 0.001% (w/v) to 20%(w/v). The amount of the electrontransport system containing the electron donor, such as NADPH, or of thepresent protein (A) may vary, for example, depending on reaction timeperiod. The reaction temperature is chosen from the range of typicallyfrom about 10° C. to 70° C., and is preferably about 20° C. to 50° C.The pH of the reaction solution is chosen from the range of typicallyfrom about 4 to 12 and is preferably about 5 to 10. The reaction timeperiod may vary as desired, and is typically from about 1 hour to 10days.

Further, the reaction in which compound (I) is reacted with the presentprotein (A) can be conducted in a cell comprising the present DNA (A).As the cells comprising the present DNA (A), for example, there ismentioned a microorganism having the ability to express the present DNA(A) and produce the present protein (A), such as, a strain of thosemicroorganisms isolated from nature comprising the present DNA (A), amutant strain derived from the microorganism strain by treatment withchemicals or ultraviolet rays, a transformed microorganism cell in whichthe present DNA (A) or a vector containing the present DNA (A) isintroduced into a host cell. Further, there is mentioned a transformedplant cell to which the present DNA (A) is introduced or a cell of atransformed plant to which the present DNA (A) is introduced. In suchcases, the compound of formula (I) may be directly applied to a cellcomprising the present DNA (A) or may be added to the culturing mediumof the cell or the soil coming into contact with the cell, so as toenter the cell. The electron transport system containing the electrondonor, such as NADPH, can be the system originally present in the celland can be added from outside of the cell.

The metabolism of compound (I) by the present protein (A) can beconfirmed, for example, by detecting the compound produced by themetabolism of compound (I). Specifically for example, compound (III)produced from metabolizing compound (II) can be detected with the HPLCanalysis or TLC analysis, described above.

Further, the metabolism of compound (I) by the present protein (A) canbe confirmed on the basis that the herbicidal activity in the reactionsolution after compound (I) is reacted with the present protein (A) iscomparatively lower than the case in which compound (I) is not reactedwith the present protein (A). As a method of testing the herbicidalactivity, for example, there is mentioned a method in which the abovereaction solutions are applied onto weeds such as barniyardgrass(Echinochloa cruis-galli), Blackgrass (Alopercurus myosuroides), Ivyleafmorningglory (Ipomoea hederacea) and Velvetleaf (Abutilon theophrasti),and the herbicidal effects are examined; or a method in which the weedsare cultivated on soil samples to which the above reaction solutions areapplied and the herbicidal effects are examined; and the like. Further,there is mentioned a method in which the above reaction solutions may bespotted onto a leaf disk taken from a plant and the presence of plantdamage (whitening) caused by the reaction solution is examined.

Further, the metabolism of compound (I) by the present protein (A) canbe confirmed by detecting as a marker, the PPO inhibitory activity inthe reaction solution after compound (I) is reacted with the presentprotein (A), which is comparatively lower than the activity in thereaction solution in which compound (1) is not reacted with the presentprotein (A). PPO is an enzyme catalyzing the conversion ofprotoporphyrinogen IX to protoporphyrin IX (hereinafter referred to as“PPIX”). For example, after adding the above reaction solutions to areaction system of PPO, protoporphyrinogen IX, which is a substrate ofPPO, is added and incubated for about 1 to 2 hours at 30° C. in thedark. Subsequently, the amount of PPIX in each of the incubatedsolutions is measured, utilizing an HPLC or the like. When the amount ofPPIX in system to which the reaction solution after compound (I) isreacted with the present protein (A) is added is more than the amount ofPPIX in system to which the reaction solution in which compound (I) isnot reacted with the present protein (A) is added, it is determined thatcompound (I) had been metabolized by the present protein (A). As PPO,there may be utilized a protein purified from plants and the like orchloroplast fraction extracted from a plant. When utilizing thechloroplast fractions, aminolevulinic acid may be utilized in thereaction system of PPO, instead of protoporphyrinogen IX. Aminolevulinicacid is the precursor of protoporphyrinogen IX in the chlorophyll-hemebiosynthesis pathway. A more specific example is given in Example 42below.

By reacting with the present protein (A) in such a way, there can beconducted a treatment of the PPO inhibitory-type herbicidal compound offormula (I), which results in metabolization and conversion of thecompound to a compound of lower herbicidal activity. The plant damagefrom said compound can be reduced by the treatment in which saidcompound which was sprayed onto the cultivation area of a plant,specifically for example, the compound which was sprayed onto thecultivation area of a plant and remains in plant residue or the soil orthe like, is reacted with the present protein (A).

As the “electron transport system containing the electron donor” whichcan be utilized to react compound (r) with the present protein (A), forexample, there can be mentioned a system containing NADPH, ferredoxinand ferredoxin-NADP⁺ reductase.

As a method of presenting the “electron transport system containing anelectron donor” in a system for reacting compound (I) with the presentprotein (A), for example, there is mentioned a method of adding to theabove reaction system, NADPH, ferredoxin derived from a plant such asspinach and ferredoxin-NADP⁺ reductase derived from a plant such asspinach. Further, there may be added to said reaction system, a fractioncontaining a component functional for the electron transport system inthe reaction system of the present protein (A), which may be preparedfrom a microorganism such as E. coli. In order to prepare such afraction, for example, after cells are harvested from a culture of amicroorganism by centrifugation or the like, the cells are disruptedphysically by an ultrasonic treatment, a DYNOMILL treatment, a FRENCHPRESS treatment and the like, or disrupted chemically by utilizing asurfactant or a cell-lyzing enzyme such as lysozyme. From the resultantlysate thus obtained, insoluble materials are removed by centrifugation,membrane filtration or the like to prepare a cell-free extract. Thecell-free extract as is can be utilized in exchange of the aboveferredoxin as the fraction containing a component functional for theelectron transport system in the reaction system of the present protein(A). Further, when a system which can transport an electron from anelectron donor to the present protein (A) is present in such a cell, aswith the case in which the reaction of the present protein (A) withcompound (I) is conducted in a cell such as a microorganism or a plantcell, no electron transport system may be newly added.

As the ferredoxin, for example, there can be utilized a ferredoxinderived from microorganisms belonging to Streptomyces, such asStreptomyces phaeochromogenes, Streptomyces testaceus, Streptomycesachromogenes, Streptomyces griseolus, Streptomyces thermocoerulescens,Streptomyces nogalater, Streptomyces tsusimaensis, Streptomycesglomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus,Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis,Streptomyces pallidus, Streptomyces roseorubens, Streptomycesrutgersensis and Streptomyces steffisburgensis, and more specifically,Streptomyces phaeochromogenes IFO 12898, Streptomyces testaceusATCC21469, Streptomyces achromogenes IFO 12735, Streptomyces griseolusATCC11796, Streptomyces thermocoerulescens IFO 14273t, Streptomycesnogalater IFO 13445, Streptomyces tsusimaensis IFO 13782, Streptomycesglomerochromogenes IFO 13673t, Streptomyces olivochromogenes IFO 12444,Streptomyces ornatus IFO 13069t, Streptomyces griseus ATCC 10137,Streptomyces griseus IFO 13849T, Streptomyces lanatus IFO 12787T,Streptomyces misawanensis IFO 13855T, Streptomyces pallidus IFO 13434T,Streptomyces roseorubens IFO 13682T, Streptomyces rutgersensis IFO15875T and Streptomyces steffisburgensis IFO 13446T, and the like; ormicroorganisms belonging to Saccharopolyspora, such as Saccharopolysporataberi, more specifically, Saccharopolyspora taberi JCM 9383t and thelike (hereinafter, sometimes collectively referred to as the “presentprotein (B)”). Specifically for example, there can be mentioned aferredoxin selected from the protein group below (hereinafter, sometimesreferred to as the “present invention protein (B)”).

Protein Group

-   (B1) a protein comprising an amino acid sequence shown in SEQ ID NO:    12 (hereinafter, sometimes referred to as the “present invention    protein (B1)”);-   (B2) a protein comprising an amino acid sequence shown in SEQ ID NO:    13 (hereinafter, sometimes referred to as the “present invention    protein (B2)”);-   (B3) a protein comprising an amino acid sequence shown in SEQ ID NO,    14 (hereinafter, sometimes referred to as the “present invention    protein (B3)”);-   (B4) a protein comprising an amino acid sequence shown in SEQ ID NO:    111 (hereinafter, sometimes referred to as the “present invention    protein (34)”);-   (B5) a ferredoxin comprising an amino acid sequence having at least    80% sequence identity with an amino acid sequence shown in any one    of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO 14 or SEQ ID NO: 111;-   (B6) a ferredoxin comprising an amino acid sequence encoded by a    nucleotide sequence having at least 90% sequence identity with a    nucleotide sequence encoding an amino acid sequence shown in any one    of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO 14 or SEQ ID NO:111;-   (B7) a protein comprising an amino acid sequence shown in SEQ ID NO:    149 (hereinafter, sometimes referred to as the “present invention    protein (B7)”);-   (B8) a protein comprising an amino acid sequence shown in SEQ ID NO:    150 (hereinafter, sometimes referred to as the “present invention    protein ([8)”);-   (B9) a protein comprising an amino acid sequence shown in SEQ ID NO:    151 (hereinafter, sometimes referred to as the “present invention    protein (B9)”);-   (B10) a protein comprising an amino acid sequence shown in SEQ ID    NO: 152 (hereinafter, sometimes referred to as the “present    invention protein (B10)”);-   (B11) a protein comprising an amino acid sequence shown in SEQ ID    NO: 153 (hereinafter, sometimes referred to as the “present    invention protein (B11)”);-   (B12) a ferredoxin comprising an amino acid sequence having at least    80% sequence identity with any one of the amino acid sequence shown    in SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153,    SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ    ID NO: 250, SEQ ID NO: 251, or SEQ ID NO: 253 or an amino acid    sequence having at least 90% sequence identity with any one of the    amino acid sequence shown in SEQ ID NO: 150, SEQ ID NO: 252 or SEQ    ID NO: 254;-   (B13) a ferredoxin comprising an amino acid sequence encoded by a    nucleotide sequence having at least 90% sequence identity with any    of the nucleotide sequence encoding an amino acid sequence shown in    SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ    ID NO: 153, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID    NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO:    253 or SEQ ID NO: 254;-   (B14) a protein comprising the amino acid sequence shown in SEQ ID    NO: 245;-   (B15) a protein comprising the amino acid sequence shown in SEQ ID    NO: 247;-   (B16) a protein comprising the amino acid sequence shown in SEQ ID    NO: 248;-   (B17) a protein comprising the amino acid sequence shown in SEQ ID    NO: 249;-   (B18) a protein comprising the amino acid sequence shown in SEQ ID    NO: 250;-   (B19) a protein comprising the amino acid sequence shown in SEQ ID    NO: 251;-   (B20) a protein comprising the amino acid sequence shown in SEQ ID    NO: 252;-   (B21) a protein comprising the amino acid sequence shown in SEQ ID    NO: 253; and-   (B22) a protein comprising the amino acid sequence shown in SEQ ID    NO: 254.

A DNA encoding the present protein (B) (hereinafter, sometimes referredto as the “present DNA (B)”) can be obtained according to conventionalgenetic engineering methods described in Molecular Cloning: A LaboratoryManual 2nd edition (1989), Cold Spring Harbor Laboratory Press; CurrentProtocols in Molecular Biology (1987), John Wiley & Sons, Incorporatedand the like, based on the nucleotide sequences encoding the amino acidsequences of the present invention protein (B) shown in SEQ ID NO: 12,13, 14, 111, 149, 150, 151, 152, 153, 245, 247, 248, 249, 250, 251, 252,253 or 254.

As the DNA encoding the present invention protein (B) (hereinafter,sometimes collectively referred to as the “present invention DNA (B)”),there is mentioned

-   -   a DNA encoding a protein comprising an amino acid sequence shown        in SEQ ID NO: 12 hereinafter, sometimes referred to as the        “present invention DNA (B1)”);    -   a DNA encoding a protein comprising an amino acid sequence shown        in SEQ E) NO: 13 (hereinafter, sometimes referred to as the        “present invention DNA (B2)”);    -   a DNA encoding a protein comprising an amino acid sequence shown        in SEQ ID NO: 14 (hereinafter, sometimes referred to as the        “present invention DNA (B3)”);    -   a DNA encoding a protein comprising an amino acid sequence shown        in SEQ ID NO: 111 (hereinafter, sometimes referred to as the        “present invention DNA (B4)”);    -   a DNA encoding a ferredoxin comprising an amino acid sequence        having at least 80% sequence identity with an amino acid        sequence shown in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ        ID NO 14 or SEQ ID NO: 111;    -   a DNA encoding a ferredoxin comprising an amino acid sequence        encoded by a nucleotide sequence having at least 90% sequence        identity with a nucleotide sequence encoding an amino acid        sequence shown in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ        ID NO 14 or SEQ ID NO: 111;    -   a DNA encoding a protein comprising an amino acid sequence shown        in SEQ ID NO: 149 (hereinafter, sometimes referred to as the        “present invention DNA (B7)”);    -   a DNA encoding a protein comprising an amino acid sequence shown        in SEQ ID NO: 150 (hereinafter, sometimes referred to as the        “present invention DNA (B8)”);    -   a DNA encoding a protein comprising an amino acid sequence shown        in SEQ ID NO: 151 (hereinafter, sometimes referred to as the        “present invention DNA (B9)”);    -   a DNA encoding a protein comprising an amino acid sequence shown        in SEQ ID NO: 152 (hereinafter, sometimes referred to as the        “present invention DNA (B10)”);    -   a DNA encoding a protein comprising an amino acid sequence shown        in SEQ ID NO: 153 (hereinafter, sometimes referred to as the        “present invention DNA (B11)”);    -   a DNA encoding a ferredoxin comprising an amino acid sequence        having at least 80% sequence identity with an amino acid        sequence shown in any one of SEQ ID NO: 149, SEQ ID NO: 151, SEQ        ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 245, SEQ ID NO: 247, SEQ        ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, or        SEQ ID NO: 253 or an amino acid sequence having at least 90%        sequence identity with an amino acid sequence shown in any one        of SEQ ID NO: 150, SEQ ID NO: 252 or SEQ ID NO 254;    -   a DNA encoding a ferredoxin comprising an amino acid sequence        encoded by a nucleotide sequence having at least 90% sequence        identity with a nucleotide sequence encoding an amino acid        sequence shown in any one of SEQ ID NO: 149, SEQ ID NO: 150, SEQ        ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 245, SEQ        ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ        ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253 or SEQ ID NO: 254;    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 245;    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 247;    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 248;    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 249;    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 250;    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 251;    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 252;    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 253; and    -   a DNA encoding a protein comprising the amino acid sequence        shown in SEQ ID NO: 254.

As more specific examples of the present invention DNA (B), there can bementioned a DNA comprising a nucleotide sequence shown in any one of SEQID NO: 15, 16, 17, 112, 154, 155, 156, 157, 158, 255, 257, 258, 259,260, 261, 262, 263 or 264, or a DNA comprising a nucleotide sequencehaving at least 90% sequence identity with a nucleotide sequence shownin any one of SEQ ID NO: 15, 16, 17, 112, 154, 155, 156, 157, 158, 255,257, 258, 259, 260, 261, 262, 263 or 264.

Such DNA can be prepared by conducting methods in which PCR is conductedwith DNA comprising a partial nucleotide sequence of the nucleotidesequences thereof as primers or hybridization methods in which such DNAis used as probes, according to the conditions described above in themethods of preparing the present DNA (A).

Specifically for example, a DNA comprising a nucleotide sequenceencoding the amino acid sequence shown in SEQ ID NO: 12 or a DNAcomprising the nucleotide sequence shown in SEQ ID NO: 15, can beprepared by conducting PCR by utilizing as the template the chromosomalDNA or chromosomal DNA library prepared from Streptomycesphaeochromogenes IFO12898 and by utilizing as primers an oligonucleotidecomprising the nucleotide sequence shown in SEQ ID NO: 105 and anoligonucleotide comprising the nucleotide sequence shown in SEQ ID NO:53.

Further, a DNA comprising a nucleotide sequence encoding the amino acidsequence shown in SEQ ID NO: 13 or a DNA comprising the nucleotidesequence shown in SEQ ID NO: 16, can be prepared by conducting PCR byutilizing as the template the chromosomal DNA or chromosomal DNA libraryprepared from Saccharopolyspora taberi JCM 9383t and by utilizing asprimers an oligonucleotide comprising the nucleotide sequence shown inSEQ ID NO: 106 and an oligonucleotide comprising the nucleotide sequenceshown in SEQ ID NO: 63.

Further, a DNA comprising a nucleotide sequence encoding the amino acidsequence shown in SEQ ID NO: 14 or a DNA comprising the nucleotidesequence shown in SEQ ID NOW 17, can be prepared by conducting PCR byutilizing as the template the chromosomal DNA or chromosomal DNA libraryprepared from Streptomyces testaceus ATCC21469 and by utilizing asprimers an oligonucleotide comprising the nucleotide sequence shown inSEQ ID NO: 107 and an oligonucleotide comprising the nucleotide sequenceshown in SEQ ID NO: 72.

Further, for example, the present invention DNA (B) can be obtained byhybridizing with a chromosomal DNA library, a DNA consisting of about atleast 20 nucleotides comprising the nucleotides sequence encoding anamino acid sequences shown in any one of SEQ ID NO: 12, 13, 14, 111,149, 150, 151, 152 or 153, as a probe under the conditions describedabove, followed by detecting and recovering the DNA which boundspecifically with said probe. The chromosomal DNA library can beprepared as described above from microorganisms belonging toStreptomyces, such as Streptomyces phaeochromogenes, Streptomycestestaceus, Streptomyces achromogenes, Streptomyces thermocoerulescens,Streptomyces nogalater, Streptomyces tsusimaensis, Streptomycesglomerochromogenes, Streptomyces olivochromogenes, Streptomyces ornatus,Streptomyces griseus, Streptomyces lanatus, Streptomyces misawanensis,Streptomyces pallidus, Streptomyces roseorubens, Streptomycesrutgersensis and Streptomyces steffisburgensis, and more specifically,Streptomyces phaeochromogenes IFO12898, Streptomyces testaceusATCC21469, Streptomyces achromogenes IFO 12735, Streptomycesthermocoerulescens IFO 14273t, Streptomyces nogalater IFO 13445,Streptomyces tsusimaensis IFO 13782, Streptomyces glomerochromogenes IFO13673t, Streptomyces olivochromogenes IFO 12444, Streptomyces ornatusIFO 13069t, Streptomyces griseus ATCC 10137, Streptomyces griseus IFO13849T, Streptomyces lanatus IFO 12787T, Streptomyces misawanensis IFO13855T, Streptomyces pallidus IFO 13434T, Streptomyces roseorubens IFO13682T, Streptomyces rutgersensis IFO 15875T and Streptomycessteffisburgensis IFO 13446T, and the like; or microorganisms belongingto Saccharopolyspora, such as Saccharopolyspora taberi, morespecifically, Saccharopolyspora taberi JCM 9383t and the like. Asspecific examples of the DNA which can be utilized as such probes, thereis mentioned a DNA comprising a nucleotide sequence shown in any one ofSEQ ID NO: 15, 16, 17, 112, 154, 155, 156, 157, 158, 255, 257, 258, 259,260, 261, 262, 263 or 264; DNA comprising a partial nucleotide sequenceof such nucleotide sequences; or a DNA comprising a nucleotide sequencecomplimentary to said partial nucleotides sequences.

To express the present DNA (B) with a host cell, for example, a DNA inwhich the present DNA (B) and a promoter fictional in a host cell areoperably linked is prepared according to conventional geneticengineering methods described in “Molecular Cloning: A Laboratory Manual2nd edition (1989)”, Cold Spring Harbor Laboratory Press; “CurrentProtocols in Molecular Biology (1987)”, John Wiley & Sons, Incorporatedand the like, and is introduced into a host cell. Whether the obtainedtransformant contains the present DNA (B) can be confirmed by preparingthe DNA from the transformant and then conducting with the prepared DNAgenetic engineering analysis methods described in, for example,“Molecular Cloning 2nd edition”, Cold Spring Harbor Press (MolecularBiology, John Wiley & Sons, N.Y. (1989) (such as confirming restrictionenzyme sites, DNA sequencing, southern hybridizations, PCR and thelike).

The present DNA (B) and the present DNA (A) can be expressed in the samecell, by introducing into a cell comprising the present DNA (A), the DNAin which the present DNA (B) and a promoter functional in a host cellare operably linked.

The present protein (B) can be prepared, for example, by culturing acell comprising the present DNA (B). As such a cell, there is mentioneda microorganism expressing the present DNA (B) and having the ability toproduce the present protein (B), such as microorganism strain isolatedfrom nature comprising the present DNA (B), mutant strains derived fromsaid natural strain by treatment with agents or ultraviolet rays or thelike. For example, there is mentioned microorganisms belonging toStreptomyces, such as Streptomyces phaeochromogenes, Streptomycestestaceus, Streptomyces achromogenes, Streptomyces griseolus,Streptomyces thermocoerulescens, Streptomyces nogalater, Streptomycestsusimaensis, Streptomyces glomerochromogenes, Streptomycesolivochromogenes, Streptomyces ornatus, Streptomyces griseus,Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus,Streptomyces roseorubens, Streptomyces rutgersensis and Streptomycessteffisburgensis, and more specifically, Streptomyces phaeochromogenesIFO 12898, Streptomyces testaceus ATCC21469, Streptomyces achromogenesIFO 12735, Streptomyces griseolus ATCC11796, Streptomycesthermocoerulescens IFO 14273t, Streptomyces nogalater IFO 13445,Streptomyces tsusimaensis IFO 13782, Streptomyces glomerochromogenes IFO13673t, Streptomyces olivochromogenes IFO 12444, Streptomyces ornatusIFO 13069t, Streptomyces griseus ATCC 10137, Streptomyces griseus IFO13849T, Streptomyces lanatus IFO 12787T, Streptomyces misawanensis IFO13855T, Streptomyces pallidus IFO 13434T, Streptomyces roseorubens IFO13682T, Streptomyces rutgersensis IFO 15875T and Streptomycessteffisburgensis IFO 13446T, and the like; or microorganisms belongingto Saccharopolyspora, such as Saccharopolyspora taberi, morespecifically, Saccharopolyspora taberi JCM 9383t and the like. Further,there can be mentioned a transformant in which the present DNA (B) hasbeen introduced. Specifically for example, there is mentioned atransformant in which the present DNA (B) operably linked to a tacpromoter, trc promoter, lac promoter or T7 phage promoter has beenintroduced into E. coli. As more specific examples, there is mentionedE. coli JM109/pKSN657FD, E. coli JM109/pKSN923FD, E. coliJM109/pKSN671FD and the like described in the examples described below.

The microorganism comprising the present DNA (B) can be cultivated inaccordance with a method employed usually to culture a microorganism,and more specifically, conducted according to the conditions describedabove in the methods of culturing the microorganism comprising thepresent DNA (A).

The present protein (B) produced by the microorganism comprising thepresent DNA (B), for example, can be utilized in various forms inreaction system of the present protein (A), such as a culture of amicroorganism producing the present protein (B), a cell of amicroorganism producing the present protein (B), a material obtained bytreating such a cell, a cell-free extract of a microorganism, a crudelypurified protein, a purified protein and the like. A material obtainedby treating a cell described above includes for example a lyophilizedcell, an acetone-dried cell, a ground cell, an autolysate of a cell, anultrasonically treated cell, an alkali-treated cell, an organicsolvent-treated cell and the like. Alternatively, the present protein(B) in any of the various forms described above may be immobilized inaccordance with known methods such as a support binding method employingan adsorption onto a synthesized polymer and the like, and an inclusionmethod employing an inclusion into a network matrix of a polymer, andthen used in the reaction system of the present protein (A).

As methods of purifying the present protein (B) from a culture of amicroorganism comprising the present DNA (B), there can be employedconventional methods utilized in a purification of protein. For example,there can be mentioned the following method.

First, cells are harvested from a culture of a microorganism bycentrifugation or the like, and then disrupted physically by anultrasonic treatment and the like, or disrupted chemically by utilizinga surfactant or a cell-lyzing enzyme such as lysozyme. From theresultant lysate thus obtained, insoluble materials are removed bycentrifugation, membrane filtration or the like to prepare a cell-freeextract, which is then fractionated by any appropriate means forseparation and purification, such as a cation exchange chromatography,an anion exchange chromatography, a hydrophobic chromatography, a gelfiltration chromatography and the like, whereby purifying the presentprotein (B). By separation of the fraction thus obtained with anSDS-PAGE, the present protein (B) can be further purified.

The function of the present protein (B) as ferredoxin can be confirmedas a function of electron transporter from ferredoxin-NADP⁺ reductase tothe present protein (A) in the reaction system in which compound (I) isreacted with the present protein (A). Specifically for example, therecan be a confirmation by adding the present protein (B) with NADPH,ferredoxin-NADP⁺ reductase and the present protein (A) to the reactionsystem in which compound (I) is reacted with the present protein (A),followed by detecting the conversion of compound (II) to compound (III).

In the method of controlling weeds of the present invention, compound(I) is applied to the cultivation area of a plant expressing the presentprotein (A). Such a plant may express one variation of the presentprotein (A) or may express multiple variations of the present protein(A). As the present protein (A), for example, there may be mentioned thepresent invention protein (A). Plants expressing the present protein (A)can be obtained as a transgenic plant to which the present DNA (A) hasbeen introduced. Such introduction involves introducing the present DNA(A) into a plant cell in the way described above so that the DNA isplaced in a position enabling its expression, followed by regenerating aplant from the obtained transformed cell. The present DNA (A) introducedinto the plant cell may have linked upstream therefrom, a nucleotidesequence encoding a transit signal to an intracellular organelle, sothat the reading frames are in frame.

The plant having introduced therein the present DNA (A) and expressingthe present protein (A) metabolizes compound (I), within its cells, intoa compound of lower herbicidal activity. As a result, the plant damagefrom the herbicidal compound in the plant is reduced and resistance tosaid compound is conferred. As such, the plant having introduced thereinthe present DNA (A) and expressing the present protein (A) can grow welleven in a case in which compound (I) is applied to a cultivation areathereof. Weeds other than the plant having introduced therein thepresent DNA (A) and expressing the present protein (A) can be removedeffectively by cultivating said plant and applying the above herbicidalcomposition to the cultivation area. It is possible to improve the yieldof the above plant, improve the quality, reduce the amount of utilizedherbicide and save labor.

The evaluation of resistance of the cell expressing the present protein(A) to the compound of formula (I) or a herbicidal compositioncomprising said compound can be carried out by contacting the cellexpressing the gene encoding the present protein (A) with said compoundor said herbicidal composition and evaluating the degree of damage tothe cell.

Specifically, to evaluate the resistance of a microorganism cellexpressing the present protein (A) to compound (I) or the herbicidalcomposition comprising compound (I), a transformed E coli expressingplant PPO and the present protein (A) may be prepared. Such preparationinvolves additionally introducing the present DNA (A) into, for example,a transformed E. coli which can be utilized to evaluate PPO activityinhibition and has been described in Japanese patent application No.11-102534, more specifically, a transformed E. coli in which a plant PPOgene described in U.S. Pat. No. 5,939,602 or the like is operablyintroduced into the E. coli BT3 strain and expressing the PPO gene. TheE. coli BT3 strain has a defect in PPO gene and has no proliferationability, as described in F. Yamamoto, H. Inokuti, H. Ozaki, (1988)Japanese Journal of Genetics, Vol. 63, pg. 237-249. The resistance tothe compound or the herbicidal composition can be evaluated bycultivating the resulting transformed E. coli with shaking for about 18to 24 hours at 37° C. in a liquid culture medium containing compound (I)or the herbicidal composition comprising said compound in an amount offrom 0 to 1.0 ppm and measuring the proliferation of said transformed E.coli with an optical density at 600 nm. As the present protein (A), forexample, there can be mentioned the present invention protein (A).

As a method of evaluating the degree of resistance of a plant expressingthe present protein (A) to the compound of formula (I) or a herbicidalcomposition comprising said compound, there is mentioned a method ofapplying the herbicidal composition to the plant and measuring thedegree of growth of the plant. For more quantitative confirmation, forexample, first, pieces of leaves of the plant are dipped in aqueoussolutions containing compound (I) at various concentrations, or theaqueous solutions of compound (I) are sprayed on pieces of leaves of theplant, followed by allowing to stand on an agar medium in the light atroom temperature. After several days, chlorophyll is extracted from theplant leaves according to the method described by Mackenney, G., J.Biol. Chem., 140; p 315 (1941) to determine the content of chlorophyll.Specifically for example, leaves of the plant are taken and are splitequally into 2 pieces along the main vein. The herbicidal composition isspread onto the full surface of one of the leaf pieces. The other leafpiece is left untreated. These leaf pieces are placed on MS mediumcontaining 0.8% agar and allowed to stand in the light at roomtemperature for 7 days. Then, each leaf piece is ground with pestle andmortar in 5 ml of 80% aqueous acetone solution to extract chlorophyll.The extract liquid is diluted 10 fold with 80% aqueous acetone solutionand the absorbance is measured at 750 nm, 663 nm and 645 nm to calculatetotal chlorophyll content according to the method described by MackenneyG., S. Biol. Chem. (1941) 140, p 315. The degree of resistance tocompound (I) can be comparatively evaluated by showing in percentilesthe total chlorophyll content of the treated leaf piece with the totalchlorophyll content of the untreated leaf piece. As the present protein(A), for example, the present invention protein (A) can be mentioned.

Based on the above method of evaluating the degree of resistance tocompound (I) or a herbicidal composition comprising compound (I), therecan be selected a plant or a plant cell showing a resistance to compound(I) or a herbicidal composition comprising compound (I). For example,there is selected a plant where no damage can be seen from sprayingcompound (I) or a herbicidal composition comprising the compound to thecultivation area of the plant, or plant cell that continuously growsthrough culturing in the presence of compound (I). Specifically, forexample, soil is packed into a plastic pot having, for example, adiameter of 10 cm and a depth of 10 cm. Seeds of the plant are sowed andcultivated in a greenhouse. An emulsion is prepared by mixing 5 parts ofa herbicidal composition comprising compound (I), 6 parts of sorpol3005X(Toho chemicals) and 89 parts of xylene. A certain amount thereof wasdiluted with water containing 0.1% (v/v) of a sticking agent at aproportion of 1000 L for 1 hectare and is spread uniformly with aspray-gun onto the all sides of the foliage from above the plantcultivated in the above pot. After cultivating the plants for 16 days ina greenhouse, the damage to the plants is investigated. The plants inwhich the damage is not observed or the plants in which the damage isreduced may be selected. Further, progeny plants can be obtained bymating such selected plants.

EXAMPLES

The present invention is explained in more detail with the Examplesbelow, but the present invention is not limited to such examples.

The HPLC for content analysis in Examples 1, 41 and 42 and fractionpurification of the compound was conducted under the conditions shownbelow.

(HPLC Analysis Condition 1)

-   column: SUMIPAX ODS211 (Sumika Chemical Analysis Service)-   column temperature: 35° C.-   flow rate: 1 ml/minute-   detection wave length: UV254 nm-   eluent A: 0.01% TFA aqueous solution-   eluent B: acetonitrile-   elution conditions: The sample is injected to the column    equilibrated with a solvent mixture of 90% of eluent A and 10%    eluent B. The solvent mixture of 900% of eluent A and 10% eluent B    is then flowed for 5 minutes. This is followed by flowing a solvent    mixture of eluent A and eluent B for 20 minutes, while increasing    the proportion of eluent B from 10% to 90%. A solvent mixture of 10%    of eluent A and 90% of eluent B is then flowed for 8 minutes.

Example 1 The Metabolism of Compound (II) by a Microorganism

(I) Metabolism of Compound (II)

The various microorganisms shown in Tables 1 and 2 were grown in ISP2agar medium (1.0%(w/v) malt extract, 0.4%(w/v) yeast extract, 0.4% (w/v)glucose, 2.0%(w/v) agar, pH 7.3). A “loopful” of the each microorganismwas added to TGY medium (0.5%(w/v) tryptone, 0.5%(w/v) yeast extract,0.1%(w/v) glucose, 0.01%(w/v) KH₂PO₄, pH 7.0) and incubated with shakingat 30° C. for 2 to 4 days. One-tenth milliliter (0.1 ml) of the obtainedculture was incubated with shaking in 3 ml of sporulation medium(0.1%(w/v) of meat extract, 0.2%(w/v) tryptose, 1% glucose, pH 7.1)containing compound (II) at 100 ppm for 7 to 8 days at 30° C. Fiftymicroliters (50 μl) of 2N HCl was added to the resulting culture andthis was exacted with 3 ml of ethyl acetate. The obtained ethyl acetatelayer was analyzed on the HPLC. The concentration of compound (II) wasreduced (column retention time of 23.9 minutes) and new peaks weredetected for compounds at retention times of 21.6 minutes and 22.2minutes (each referred to as metabolite (I) and metabolite (II)). Theresults are shown in Tables 1 and 2. TABLE 1 concentration peak area ofpeak area of strain of the of compound metabolite (I) metabolite (II)microorganism (II) (ppm) (×10⁴) (×10⁴) Streptomyces 77.8 3.43 3.57cacaoiasoensis IFO13813 Streptomyces 49.5 7.96 9.86 griseofuscusIFO12870t Streptomyces 65.3 4.30 5.00 ornatus IFO13069t Streptomyces51.7 7.47 9.16 thermocoerulescens IFO14273t Streptomyces 81.9 0.71 0.82roseochromogenes ATCC13400 Streptomyces 89.6 1.02 1.50 lavendulaeATCC11924 Streptomyces 65.6 6.19 1.30 griseus ATCC10137 Streptomyces30.3 12.8 156 griseus ATCC11429 Streptomyces 51.1 0.52 2.27 griseusATCC12475 Streptomyces 75.2 1.91 2.26 griseus ATCC15395 Streptomyces54.6 4.94 6.05 erythreus ATCC11635 Streptomyces 88.3 3.28 4.40 scabiesIFO3111 Streptomyces 22.6 14.4 18.5 griseus IF03102 Streptomyces 85.33.81 1.59 catenulae IFO12848 Streptomyces 92.4 1.08 0.91 kasugaensisATCC15714 Streptomyces 70.9 2.30 2.87 rimosus ATCC10970 Streptomyces 0.015.9 21.8 achromogenes IFO12735 Streptomyces 62.0 5.48 6.69 lydicusIFO13058

TABLE 2 concentration peak area of peak area of strain of the ofcompound metabolite (I) metabolite (II) microorganism (II) (ppm) (×10⁴)(×10⁴) Streptomyces 46.4 8.28 10.5 phaeochromogenes IFO12898Streptomyces 80.6 2.54 3.59 afghaniensis IFO12831 Streptomyces 83.9 4.992.91 hachijoensis IFO12782 Streptomyces 13.0 14.9 19.2 argenteolus var.toyonakensis ATCC21468 Streptomyces 18.4 11.6 14.4 testaceus ATCC21469Streptomyces 70.9 5.37 6.11 purpurascens ATCC25489 Streptomyces 53.93.00 3.97 griseochromogenes ATCC14511 Streptomyces 66.3 12.1 12.6kasugaensis IFO13851 Streptomyces 90.1 2.75 3.01 argenteolus var. toyonATCC21468t Streptomyces 71.8 4.66 4.00 roseochromogenes ATCC13400tStreptomyces 12.8 21.9 24.9 nogalater IFO13445 Streptomyces 74.2 4.145.87 roseochromogenus ATCC21895 Streptomyces 46.5 8.33 11.3 fimicariusATCC21900 Streptomyces 61.1 3.70 3.94 chartreusis ATCC21901 Streptomyces79.9 2.86 2.52 globisporus subsp. globisporus ATCC21903 Streptomyces 014.4 19.9 griseolus ATCC11796 Saccharopolyspora 82.9 5.83 7.71 taberiJCM9383T Streptomyces sp. 54.6 2.30 3.44 SANK62585(2) Structure Determination of the Metabolite (I) and Metabolite (II)

A frozen stock of Streptomyces griseus ATCC 11429 was added to 3 ml of amicroorganism culture medium (0.7%(w/v) polypeptone, 0.5%(w/v) yeastextract, 1.0%(w/v) of glucose, 0.5%(w/v) of K₂HPO₄, pH7.2) and incubatedwith shaking in a test tube overnight to obtain a pre-culture. Suchpre-culture was added to 300 ml of the microorganism medium containingcompound (II) at a concentration of 100 ppm. This was divided into 100small test tubes at 3 ml each and incubated with shaking at 30° C. for 6days. After 250 ml of such culture was adjusted to a pH2 by adding HCl,this was extracted with 250 ml of ethyl acetate. The solvents wereremoved from the ethyl acetate layer. The residue was dissolved in 3 mlof acetone and spotted to a silica gel TLC plate (TLC plate silica gel60F₂₅₄, 20 cm×20×m, 0.25 mm thickness, Merck Company). The TLC plate wasdeveloped with 5:7:1 (v/v/v) mixture of toluene, formic acid and ethylformate. The Rf value around 0.58 of the silica gel was taken. Suchcontents of the TLC plate were extracted with acetone. The acetone wasremoved from the extraction layer. The residue was dissolved in 10 ml ofacetonitrile and fractionated with a HPLC. The fractions containing onlymetabolite (I) and metabolite (II) were recovered to obtain 3.7 mg ofmetabolites (hereinafter referred to as “metabolite A”).

Mass spectrometry analysis of metabolite A was conducted. Metabolite Ahad a mass that was 14 smaller than compound (II). Further, from H-NMRanalysis, it was determined that metabolite (A) was a compound havingthe structure shown in formula (III).

(3) Herbicidal Activity Test of Compound (III)

Soil was packed into a round plastic pot having a diameter of 10 cm anddepth of 10 cm. Barnyardgrass, Blackgrass, Ivyleaf morningglory wereseeded and cultivated in a greenhouse for 10 days. Five (5) parts of thetest compound, 6 parts of sorpol3005X (Toho Chemical Company) and 89parts of xylene were well mixed to produce an emulsion. A certain amountthereof was diluted with water containing 0.1% (v/v) of a sticking agentat a proportion of 1000 L for 1 hectare and was spread uniformly with aspray-gun onto the all sides of the foliage from above the plantcultivated in the above pot. After cultivating the plants for 16 days ina greenhouse, the herbicidal activity of the test compound wasinvestigated. The results are shown in Table 3. TABLE 3 HerbicidalActivity concen- Ivyleaf test tration Barnyard- Black- Morning-compounds (g/ha) grass grass glory compound (II) 500 10 10 10 125 10 1010 compound (III) 500 0 0 0 125 0 0 0

Soil was packed into a round plastic pot having a diameter of 10 cm anddepth of 10 cm. Barnyardgrass, Blackgrass, Ivyleaf morningglory wereseeded. Five (5) parts of the test compound, 6 parts of sorpol3005X(Toho Chemical Company) and 89 parts of xylene were well mixed toproduce an emulsion. A certain amount thereof was diluted with watercontaining 0.1% (v/v) of a sticking agent at a proportion of 1000 L for1 hectare and was spread uniformly with a spray-gun onto the surface ofthe soil. After cultivating the plants for 19 days in a greenhouse, theherbicidal activity was investigated. The results are shown in Table 4.TABLE 4 Herbicidal Activity concen- Ivyleaf test tration Barnyard-Black- Morning- compounds (g/ha) grass grass glory compound (II) 500 1010 10 compound (III) 500 0 0 0

In the above Tables 3 and 4, the strength of the herbicidal activity isshown stepwise as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The number “0”represents situations in which the condition of sprouting or vegetationat the time of examination of the plant utilized for the test wascompared with and showed totally or substantially no difference withthat of the untreated application. The number “10” represents situationsin which the plant completely withered or the sprouting or vegetationwas completely suppressed.

Example 2 Preparation of the Present Invention Protein (A1)

(1) Preparation of the Crude Cell Extract

A frozen stock of Streptomyces phaeochromogenes IFO12898 was added to100 ml of A medium (0.1%(w/v) glucose, 0.5%(w/v) tryptone, 0.5%(w/v)yeast extract, 0.1%(w/v) of dipotassium hydrogenphosphate, pH7.0) in a500 ml triangular flask and incubated with rotary shaking at 30° C. for1 day to obtain a pre-culture. Eight milliliters (8 ml) of thepre-culture was added to 200 ml of A medium and was incubated withrotary shaking in 500 ml a baffled flask at 30° C. for 2 days. Cellpellets were recovered by centrifuging (3,000 g, 5 mm.) the resultingculture. These cell pellets were suspended in 100 ml of B medium (1%(w/v) glucose, 0.1% beef extract, 0.2%(w/v) tryptose) containingcompound (II) at 100 ppm and were incubated with reciprocal shaking in a500 ml Sakaguchi flask for 16 hours at 30° C. Cell pellets wererecovered by centrifuging (3,000 g, 5 min.) 10 L of the resultingculture. The resulting cell pellets were washed twice with 1 L of 0.1Mpotassium phosphate buffer (pH7.0) to provide 162 g of the cell pellets.

These cell pellets were suspended in 0.1M potassium phosphate buffer(pH7.0) at 2 ml for 1 g of the cell pellets, and 1 mM PMSF, 5 mMbenzamidine HCl, 1 mM EDTA and 1 mM of dithiotritol were added thereto.A cell lysate solution was obtained by disrupting twice repetitively thesuspension with a French press (1000 kg/cm²) (Ohtake Seisakusho). Aftercentrifuging the cell lysate solution (40,000×g, 30 minutes), thesupernatant was recovered and centrifuged for 1 hour at 150,000×g torecover the supernatant (hereinafter referred to as the “crude cellextract”).

(2) Determination of the Ability of Converting Compound (II) to Compound(III)

There was prepared 30 μl of a reaction solution of 0.1M potassiumphosphate buffer (pH7.0) containing 3 ppm of compound (II) labeled with¹⁴C, 2.4 mM of β-NADPH (hereinafter, referred to as “component A”)(Oriental Yeast Company), 0.5 mg/ml of a ferredoxin derived from spinach(hereinafter referred to as “component B”) (Sigma Company), 1 U/ml offerredoxin reductase (hereinafter, referred to as “component C”) (SigmaCompany) and 18 μl of the crude cell extract recovered in Example 2(1).The reaction solution was maintained at 30° C. for a hour. Further,there was prepared and maintained similarly a reaction solution havingno addition of at least one component utilized in the composition of theabove reaction solution, selected from component A, component B andcomponent C. Three microliters (3 μl) of 2N HCl and 90 μl of ethylacetate were added and mixed into each of the reaction solutions afterthe maintenance. The resulting reaction solutions were centrifuged at8,000×g to recover 75 μl of the ethyl acetate layer. After drying theethyl acetate layers under reduced pressure, the residue was dissolvedin 6.0 μl of ethyl acetate. Five microliters (5.0 μl) thereof wasspotted to a TLC plate (TLC plate silica gel 60F₂₅₄ 20 cm×20 cm, 0.25thick, Merck Company). The TLC plate was developed with a 6:1:2 mixtureof chloroform, acetic acid and ethyl acetate for about 1 hour. Thesolvents were then allowed to evaporate. The TLC plate was exposedovernight to an imaging plate (Fuji Film Company). Next, the imagingplate was analyzed on Image Analyzer BAS2000 (Fuji Film Company). Thepresence of a spot corresponding to compound (III) labeled with ¹⁴C wereexamined (Rf value 0.24 and 0.29). The results are shown in Table 5.TABLE 5 Reaction components component component component crude cellcompound (II) spot of A B C extract labeled with¹⁴C compound (III) + + +− + − + + + + + + − + + + + − + − − + + −(3) Fractionation of the Crude Cell Extract

Ammonium sulfate was added to the crude cell extract obtained in Example2(1) to amount to 45% saturation. After stirring in ice-cooledconditions, the supernatant was recovered by centrifugation for 10minutes at 12,000×g. After adding ammonium sulfate to the obtainedsupernatant to amount to 55% saturation and string in ice-cooledconditions, a pellet was recovered by centrifuging for 10 minutes at12,000×g. The pellet was dissolved with 27.5 ml of 20 mM bistispropanebuffer (pH7.0). This solution was subjected to a PD10 column (AmershamPharmacia Company) and eluted with 20 mM of bistrispropane buffer(pH7.0) to recover 38.5 ml of fractions containing proteins (hereinafterreferred to as the “45-55% ammonium sulfate fraction”).

(4) Isolation of the Present Invention Protein (A1)

The 45-55% ammonium sulfate fraction prepared in Example 2(3) wasinjected into a HiLoad26/10 Q Sepharose HP column (Amersham PharmaciaCompany). Next, after flowing 106 ml of 20 mM bistrispropane buffer(pH7.0) into the column, 20 mM bistrispropane buffer was flown with alinear gradient of NaCl (gradient of NaCl was 0.001415M/minute, range ofNaCl concentration was from 0M to 0.375M, flow rate was 3 ml/minute) tofraction recover 25 ml of fractions eluting at the NaCl concentration offrom 0.21M to 0.22M. Further, the recovered fractions were subjected toa PD10 column (Amersham Pharmacia Biotech Company) and eluted with 20 mMbistrispropane buffer (pH7.0) to recover the fractions containingprotein.

The recovered fractions were subjected to a PD10 column (AmershamPharmacia Biotech Company) with the elution with Buffer A (2 mMpotassium phosphate buffer containing 1.5 mM of NaCl, pH 7.0), in orderto recover the fractions containing protein. Next, the fractions wereinjected into a Bio-Scale Ceramic Hydroxyapatite Type I column CHT10-I(BioRad Company). Thirty milliliters (30 ml) of Buffer A was flown intothe column. Subsequently, Buffer A was flown with a linear gradient ofBuffer B (10 nM potassium phosphate buffer containing 0.03 mM of NaCl;the linear gradient started at 100% Buffer A to increase to 50% Buffer Bover a 100 minute period, flow rate was 2 ml/minute) to fraction recoverthe fractions eluting at a Buffer B concentration of from 17% to 20%Further, the recovered fractions were subjected to a PD10 column(Amersham Pharmacia Biotech Company) and eluted with 0.05M potassiumphosphate buffer (pH7.0) to recover the fractions containing protein.

The recovered fractions were concentrated 20 fold using an ultrafiltermembrane (Microcon YM-30, Millipore Company) and injected into a HiLoad16/60 Superdex 75 pg column (Amersham Pharmacia Biotech Company). Fiftymillimolar (50 mM) potassium phosphate buffer containing 0.15M of NaCl(pH7.0) was flown (flow rate 1 ml/minute) into the column. The elutionwas factioned at 2 ml each. The fractions eluting at the elution volumesof from 56 ml to 66 ml were each fraction recovered. The proteincontained in each of the fractions was analyzed with a 10%-20% SDS-PAGE.

Instead of the crude cell extract in the reaction solution described inExample 2(2), the recovered fractions were added and maintained in thepresence of component A, component B, component C and compound (II)labeled with ¹⁴C, similarly to Example 2(2). The reaction solutionsafter the maintenance were TLC analyzed to examine the intensity of thespots corresponding to compound (III) labeled with ¹⁴C. The proteinmoving to the position to 47 kDa in the above SDS-PAGE was observed tohave its fluctuations in the concentrations of the bands of thefractions added in turn to be parallel with the fluctuations of theintensity of the spots corresponding to compound (III). Said protein wasrecovered from the SDS-PAGE gel and was subjected to an amino acidsequence analysis with a protein sequencer (Applied Biosystems Company,Procise 494HT, pulsed liquid method). As a result, the amino acidsequence shown in SEQ ID NO: 18 was provided. Further, after digestingthe above protein with trypsin, the obtained digestion material wasanalyzed on a mass spectrometer (ThermoQuest Company, Ion Trap MassSpectrometer LCQ, column: LC Packings Company PepMap C18 75 μm×150 mm,solvent A: 0.1% HOAc—H₂O, solvent B: 0.1% HOAc-methanol, gradient: alinear gradient starting at an elution with a mixture of 95% of solventA and 5% of solvent B and increasing to a concentration of 100% ofsolvent B over 30 minutes, flow rate: 0.2 μl/minute). As a result, thesequence shown in SEQ ID NO: 19 was provided.

Example 3 Obtaining the Present Invention DNA (A1)

(1) Preparation of the Chromosomal DNA of Streptomyces phaeochromogenesIFO12898

Streptomyces phaeochromogenes IFO12898 was incubated with shaking at 30°C. for 1 day to 3 days in 50 ml of YEME medium (0.3%(w/v) yeast extract,0.5%(w/v) bacto-peptone, 0.3%(w/v) malt extract, 1.0%(w/v) glucose,34%(w/v) sucrose and 0.2%(v/v) 2.5M MgCl₂.6H₂O). The cells wererecovered. The obtained cells were suspended in YEME medium containing1.4/(w/v) glycine and 60 mM EDTA and further incubated with shakking fora day. The cells were recovered from the culture medium. After washingonce with distilled water, it was resuspended in buffer (100 mM Tris-HCl(pH8.0), 100 mM EDTA, 10 mM NaCl) at 1 ml per 200 mg of the cells. Twohundred micrograms per milliliter (200 μg/ml) of egg-white lysozyme wereadded. The cell suspension was incubated with shaking at 30° C. for ahour. Further, 0.5% of SDS and 1 mg/ml of Proteinase K was added. Thecell suspension was incubated at 55° C. for 3 hours. The cell suspensionwas extracted twice with mixture of phenol, chloroform and isoamylalcohol to recover each of the aqueous layers. Next, there was oneextraction with mixture of chloroform and isoamyl alcohol to recover theaqueous layer. The chromosomal DNA was obtained by ethanol precipitationfrom the aqueous layer.

(2) Preparation of the Chromosomal DNA Library of Streptomycesphaeochromogenes IFO12898

Nine hundred forty-three nanograms (943 ng) of the chromosomal DNAprepared in Example 3(1) were digested with 1 unit of restriction enzymeSau3AI at 37° C. for 60 minutes. The obtained digestion solution wasseparated with 0.7% agarose gel electrophoresis. The DNA of about 2.0kbp was recovered from the gel. The DNA was purified with aPrep-A-Gene^(R) DNA purification kit (Bio-Rad company) according to theinstructions attached to said kit to obtain 10 μL of the solutioncontaining the target DNA. A microliter (1 μl) of the DNA solution, 98ng of plasmid vector pUC118 digested with restriction enzyme BamHI andtreated with dephosphorylation and 11 μl of the I solution from LigationKit Ver. 2 (Takara Shuzo Company) were mixed and incubated overnight at16° C. E coli DH5α was transformed utilizing 51 μl of the ligationsolution. The E. coli was cultured with shaking overnight at 30° C. Fromthe obtained culture medium, the E. coli was recovered. The plasmid wasextracted to provide the chromosomal DNA library.

(3) Isolation of the Present Invention DNA (A1)

PCR was conducted by utilizing as the template the chromosomal DNAprepared in Example 3(1) (FIG. 1). As the primers, there was utilizedthe pairing of an oligonucleotide having the nucleotide sequence shownin SEQ ID NO: 35 and an oligonucleotide having the nucleotide sequenceshown in SEQ ID NO: 36 (hereinafter referred to as “primer paring 1”).The nucleotide sequence shown in SEQ ID NO: 35 was designed based on anucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:18. Further, the nucleotide sequence shown in SEQ ID NO: 36 was designedbased on a nucleotide sequence complimentary to the nucleotide sequenceencoding the amino acid sequence shown in SEQ ID NO: 19. The PCRreaction solution amounted to 25 μl by adding the 2 primers eachamounting to 200 nM, 250 ng of the above chromosomal DNA, 0.5 μl of dNTPmix (a mixture of 10 mM of each of the 4 types of dNTP; ClontechCompany), 5 μl of 5×GC genomic PCR reaction buffer (Clontech Company),1.1 μl of 25 mM Mg(OAc)₂, 5 μl of 5M GC-Melt (Clontech Company) and 0.5μl of Advantage-GC genomic polymerase mix (Clontech Company) anddistilled water. The reaction conditions of the PCR were aftermaintaining 95° C. for 1 minute, repeating 30 cycles of a cycle thatincluded maintaining 94° C. for 15 seconds, followed by 60° C. for 30seconds, followed by 72° C. for 1 minute, and then maintaining 72° C.for 5 minutes. After the maintenance, the reaction solution wassubjected to 4% agarose gel electophoresis. The gel area containing theDNA of about 150 bp was recovered. The DNA was purified from therecovered gel by utilizing QIAquick gel extraction kit (Qiagen Company)according to the attached instructions. The obtained DNA was ligated tothe TA cloning vector pCR2.1 (Invitrogen Company) according to theinstructions attached to said vector and was introduced into E. ColiTOP10F′. The plasmid DNA was prepared from the obtained E. colitransformant, utilizing QIAprep Spin Miniprep Kit (Qiagen Company). Asequencing reaction was conducted with Dye terminator cycle sequencingFS ready reaction kit (Applied Biosystems Japan Company) according tothe instructions attached to said kit, utilizing as primers the −21M13primer (Applied Biosystems Japan Company) and M13Rev primer (AppliedBiosystems Japan Company). The sequencing reaction utilized the obtainedplasmid DNA as the template. The reaction products were analyzed with aDNA sequencer 373A (Applied Biosystems Japan Company). As a result, thenucleotide sequence shown in nucleotides 36 to 132 of the nucleotidesequence shown in SEQ ID NO: 9 was provided. Said nucleotide sequenceencoded the amino acid sequence shown in amino acids 12 to 23 of theamino acid sequence shown in SEQ ID NO: 18. In this regard, it wasexpected that said DNA encoded a part of the present invention protein(A1).

Next, PCR was conducted similar to the above with Advantage-GC genomicpolymerase mix (Clontech Company) and by utilizing the chromosomal DNAprepared in Example 3(2) as the template. There was utilized as primers,a pairing of an oligonucleotide having the nucleotide sequence shown inSEQ ID NO: 37 with an oligonucleotide having the nucleotide sequenceshown in SEQ ID NO: 38 (hereinafter referred to as the “primer pairing2”) or a pairing of an oligonucleotide having the nucleotide sequenceshown in SEQ ID NO: 39 with an oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 40 (hereinafter referred to as the “primerpairing 3”).

Next, there was amplified by PCR a DNA having a nucleotide sequence inwhich the 3′ terminus extends past the nucleotide shown as nucleotide132 of the nucleotide sequence shown in SEQ ID NO: 9. The PCR wasconducted by utilizing as the template solution the reaction solutionobtained with the use of primer pairing 2 and by utilizing as primers apairing of the oligonucleotide having the nucleotide sequence shown inSEQ ID NO: 41 and the oligonucleotide having the nucleotide sequenceshown in SEQ ID NO: 38 hereinafter referred to as “primer pairing 4”).Similarly, there was amplified by PCR a DNA having a nucleotide sequencein which the 5′ terminus extends past the nucleotide shown as nucleotide36 of the nucleotide sequence shown in SEQ ID NO: 9. The PCR wasconducted by utilizing as the template solution the reaction solutionobtained with the use of primer pairing 3 and by utilizing as primers apairing of the oligonucleotide having the nucleotide sequence shown inSEQ ID NO: 42 and the oligonucleotide having the nucleotide sequenceshown in SEQ ID NO: 40 (hereinafter referred to as “primer pairing 5”).The 2 kbp DNA amplified with the use of primer pairing 4 and the 150 bpDNA amplified with the use of primer pairing 5 are cloned into TAcloning vector pCR2.1, similar to the above. Plasmid DNA was preparedfrom the obtained E. coli transformant, utilizing QIAprep Spin MiniprepKit (Qiagen Company). A sequencing reaction was conducted with Dyeterminator cycle sequencing FS ready reaction kit (Applied BiosystemsJapan Company) according to the instructions attached to said kit,utilizing as primers the −21M13 primer (Applied Biosystems JapanCompany), M13Rev primer (Applied Biosystems Japan Company) and theoligonucleotides shown in SEQ ID NO: 43-50. The sequencing reactionutilized the obtained plasmid DNA as the template. The reaction productswere analyzed with a DNA sequencer 373A (Applied Biosystems JapanCompany). As a result of sequencing the nucleotide sequence of the 2 kbpDNA amplified by utilizing primer pairing 4, the nucleotide sequenceshown in nucleotides 133 to 1439 of the nucleotide sequence shown in SEQID NO: 9 was provided. Further, as a result of sequencing the nucleotidesequence of the 150 bp DNA amplified by utilizing primer pairing 5, thenucleotide sequence shown in nucleotides 1 to 35 of the nucleotidesequence shown in SEQ ID NO: 9 was provided. As a result of connectingthe obtained nucleotide sequences, the nucleotide sequence shown in SEQID NO: 9 was obtained. Two open reading frames (ORF) were present insaid nucleotide sequence. As such, there was contained a nucleotidesequence (SEQ ID NO: 6) consisting of 1227 nucleotides (inclusive of thestop codon) and encoding a 408 amino acid residue as well as anucleotide sequence (SEQ ID NO: 15) consisting of 201 nucleotides(inclusive of the stop codon) and encoding a 66 amino acid residue. Themolecular weight of the protein consisting of the amino acid sequence(SEQ ID NO: 1) encoded by the nucleotide sequence shown in SEQ ID NO: 6was calculated to be 45213 Da. Further, the amino acid sequence encodedby said nucleotide sequence contained the amino acid sequence (SEQ IDNO: 18) determined from the amino acid sequencing of from the N terminusof the present invention protein (A1) and the amino acid sequence (SEQID NO: 19) determined from the amino acid sequencing of the trypsindigestion fragments with the mass spectrometer analysis. The molecularweight of the protein consisting of the amino acid sequence (SEQ ID NO:12) encoded by the nucleotide sequence shown in SEQ ID NO: 15 wascalculated to be 6818 Da.

Example 4 Expression of the Present Invention Protein (A1) in E. coli

(1) Production of a Transformed E. coli Having the Present InventionProtein (A1)

PCR was conducted by utilizing as a template the chromosomal DNAprepared from Streptomyces phaeochromogenes IFO12898 in Example 3(1) andby utilizing Expand High Fidelity PCR System (Roche MolecularBiochemicals Company). As the primers, there was utilized the paring ofan oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 51and an oligonucleotide having the nucleotide sequence shown in SEQ IDNO: 52 (hereinafter referred to as “primer pairing 19”) or a pairing ofan oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 51and an oligonucleotide having the nucleotide sequence shown in SEQ IDNO: 53 (hereinafter referred to as “primer pairing 20”). The PCRreaction solution amounted to 50 μl by adding the 2 primers eachamounting to 300 nM, 50 ng of the above chromosomal DNA, 5.0 μl of dNTPmix (a mixture of 2.0 mM of each of the 4 types of dNTP), 5.0 μl of 10×Expand HF buffer (containing MgCl₂) and 0.75 μl of Expand HiFi enzymemix and distilled water. The reaction conditions of the PCR were aftermaintaining 97° C. for 2 minutes; repeating 10 cycles of a cycle thatincluded maintaining 97° C. for 15 seconds, followed by 65° C. for 30seconds and followed by 72° C. for 2 minutes; then conducting 15 cyclesof a cycle that included maintaining 97° C. for 15 seconds, followed by68° C. for 30 seconds and followed by 72° C. for 2 minutes (wherein 20seconds was added to the maintenance at 72° C. for each cycle); and thenmaintaining 72° C. for 7 minutes. After the maintenance, the reactionsolution was subjected to 1% agarose gel electrophoresis. The gel areacontaining the DNA of about 1.2 kbp was recovered from the gel which wassubjected the reaction solution utilizing primer pairing 19. The gelarea containing the DNA of about 1.5 kbp was recovered from the gelwhich was subjected the reaction solution utilizing primer pairing 20.The DNA were purified from each of the recovered gels by utilizingQIAquick gel extraction kit (Qiagen Company) according to the attachedinstructions. The obtained DNA were ligated to the TA cloning vectorpCR2.1 (Invitrogen Company) according to the instructions attached tosaid vector and were introduced into E. Coli TOP10F′. The plasmid DNAwere prepared from the obtained E. coli transformants, utilizing QIAprepSpin Miniprep Kit (Qiagen Company). Sequencing reactions were conductedwith Dye terminator cycle sequencing FS ready reaction kit (AppliedBiosystems Japan Company) according to the instructions attached to saidkit, utilizing as primers the −21M13 primer (Applied Biosystems JapanCompany), M13Rev primer (Applied Biosystems Japan Company), theoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 43and the oligonucleotide having the nucleotide sequence shown in SEQ IDNO: 46. The sequencing reactions utilized the obtained plasmid DNA asthe template. The reaction products were analyzed with a DNA sequencer373A (Applied Biosystems Japan Company), Based on the results, theplasmid having the nucleotide sequence shown in SEQ ID NO: 6 wasdesignated as pCR657 and the plasmid having the nucleotide sequenceshown in SEQ ID NO: 9 was designated as pCR657F.

Furthermore, the oligonucleotide having the nucleotide sequence shown inSEQ ID NO: 134 and the oligonucleotide having the nucleotide sequenceshown in SEQ ID NO: 135 were annealed together to provide a linker (FIG.47). Plasmid pKSN24R2 (Akiyoshi-ShibaTa M. et al., Eur. J. Biochem. 224:P335(1994)) was digested with HindIII and XmnI. The linker was insertedinto the obtained DNA of about 3 kb. The obtained plasmid was designatedas pKSN2 (FIG. 4).

Next, each of plasmids pCR657 and pCR657F was digested with restrictionenzymes NdeI and HindIII. The digestion products were subjected toagarose gel electrophoresis. The gel area containing a DNA of about 1.2kbp was cut from the gel subjected to the digestion products of pCR657.The gel area containing a DNA of about 1.5 kbp was cut from the gelsubjected to the digestion products of pCR657F. The DNA were purifiedfrom each of the recovered gels by utilizing QIAquick gel extraction kit(Qiagen Company) according to the attached instructions. Each of theobtained DNA and the plasmid pKSN2 digested with NdeI and HindIII wereligated with ligation kit Ver.1 (Takara Shuzo Company) according to theinstructions attached to said kit and introduced into E. Coli JM109. Theplasmid DNA were prepared from the obtained E. coli transformants. Thestructures thereof were analyzed. The plasmid containing the nucleotidesequence shown in SEQ ID NO: 6, in which the DNA of about 1.2 kbpencoding the present invention protein (A1) is inserted between the NdeIsite and the HindIII site of pKSN2 was designated as pKSN657. Further,the plasmid containing the nucleotide sequence shown in SEQ ID NO: 9, inwhich the DNA of about 1.5 kbp encoding the present invention protein(A1) is inserted between the NdeI site and the HindIII site of pKSN2 wasdesignated as pKSN657F. Each of the above plasmids of pKSN657 andpKSN657F were introduced into E. coli JM109. The obtained E. colitransformants were designated, respectively, JM109/pKSN657 andJM109/pKSN657F. Further, plasmid pKSN2 was introduced into E. coliJM109. The obtained E. coli transformant was designated as JM109/pKSN2.

(2) Expression of the Present Invention Protein (A1) in E. coli andRecovery of Said Protein

E. coli JM109/pKSN657, JM109/pKSN657F and JM109/pKSN2 were each culturedovernight at 37° C. in 10 ml of TB medium (1.2%(w/v) tryptone, 2.4%(w/v)of yeast extract, 0.4%(w/v) of glycerol, 17 mM potassiumdihydrogenphosphate, 72 mM dipotassium hydrogenphosphate) containing 50μg/ml of ampicillin. A milliliter (1 ml) of the obtained culture mediumwas transferred to 100 ml of TB medium containing 50 μg/ml of ampicillinand cultured at 26° C. When OD660 reached about 0.5, 5-aminolevulinicacid was added to the final concentration of 500 μM, and the culturingwas continued. Thrity (30) minutes thereafter, IPTG was added to a finalconcentration of 1 mM, and there was further culturing for 17 hours.

The cells were recovered from each of the culture mediums, washed with0.1M tris-HCl buffer (pH7.5) and suspended in 10 ml of the above buffercontaining 1 mM PMSF. The obtained cell suspensions were subjected 6times to a sonicator (Sonifier (Branson Sonic Power Company)) at 3minutes each under the conditions of output 3, duty cycle 30%, in orderto obtain cell lysate solutions. After centrifuging the cell lysatesolutions (1,200×g, 5 minutes) the supernatants were recovered andcentrifuged (150,000×g, 70 minutes) to recover supernatant fractions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN657 is referred to as “E. coli pKSN657 extract”, thesupernatant fraction obtained from E. coli JM109/pKSN657F is referred toas “E. coli pKSN657F extract”, and the supernatant fraction obtainedfrom E. coli JM109/pKSN2 is referred to as “E. coli pKSN2 extract”). Amicroliter (1 μl) of the above supernatant fractions was analyzed on a15% to 25% SDS-PAGE and stained with Coomasie Blue (hereinafter referredto as “CBB”). As a result, notably more intense bands were detected inboth E. coli pKSN657 extract and E. coli pKSN657F extract than the E.coli pKSN2 extract, at the electrophoresis locations corresponding tothe molecular weight of 47 kDa. A more intense band was detected in E.coli pKSN657F extract than E. coli pKSN657 extract. It was shown that E.coli JM109/pKSN657F expressed the present invention protein (A1) to ahigher degree than E. coli JM109/pKSN657.

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Reaction solutions of 30 μl were prepared and maintained for 1 hour at30° C. The reaction solutions consisted of a 0.1M potassium phosphatebuffer (pH7.0) containing 3 ppm of compound (II) labeled with ¹⁴C, 2 mMof β-NADPH (hereinafter, referred to as “component A”) (Oriental YeastCompany), 0.2 mg/ml of a ferredoxin derived from spinach (hereinafterreferred to as “component B”) (Sigma Company), 1 U/ml of ferredoxinreductase (hereinafter, referred to as “component C”) (Sigma Company)and 18 μl of the supernatant fraction recovered in Example 4(2).Further, there were prepared and maintained similarly reaction solutionshaving no addition of at least one component utilized in the compositionof the above reaction solution, selected from component A, component Band component C. Three microliters (3 μl) of 2N HCl and 90 μl of ethylacetate were added and stirred into each of the reaction solutions afterthe maintenance. The resulting reaction solutions were centrifuged at8,000×g to recover 75 μl of the ethyl acetate layer. After drying theethyl acetate layers under reduced pressure, the residue was dissolvedin 6.0 μl of ethyl acetate. Five microliters (5.0 μl) thereof wasspotted to a TLC plate (TLC plate silica gel 60F₂₅₄ 20 cm×20 cm, 0.25thick, Merck Company). The TLC plate was developed with a 6:1:2 mixtureof chloroform, acetic acid and ethyl acetate for about 1 hour. Thesolvents were then allowed to evaporate. The TLC plate was exposedovernight to an imaging plate (Fuji Film Company). Next, the imagingplate was analyzed on Image Analyzer BAS2000 (Fuji Film Company). Thepresence of a spot corresponding to compound (III) labeled with ¹⁴C wereexamined (Rf value 0.24 and 0.29). The results are shown in Table 6.TABLE 6 Reaction components component component component E. colicompound (II) spot of A B C extract labeled with¹⁴ C compound (HI) + + +− + − + + + pKSN2 + − + + + pKSN657 + + − + + pKSN657 + − + − +pKSN657 + − + + − pKSN657 + + + + + pKSN657F + + − + + pKSN657F + − +− + pKSN657F + − + + − pKSN657F + +

Example 5 Preparation of the Present Invention Protein (A2)

(1) Preparation of the Crude Cell Extract

A frozen stock of Saccharopolyspora taberi JCM 9383t was added to 10 mlof A medium (0.1%(w/v) glucose, 0.5%(w/v) tryptone, 0.5%(w/v) yeastextract, 0.1%(w/v) of dipotassium hydrogenphosphate, pH7.0) in a 10 mltest tube and incubated with shaking at 30° C. for 1 day to obtain apre-culture. Eight milliliters (8 ml) of the pre-culture was added to200 ml of A medium and was revolve cultured in 500 ml a baffled flask at30° C. for 2 days. Cell pellets were recovered by centrifuging (3,000×g,10 min.) 10 L of the resulting culture. These cell pellets weresuspended in 100 ml of B medium (1%(w/v) glucose, 0.1% beef extract,0.2%(w/v) tryptose) containing compound (IX) at 100 ppm and wereincubated with reciprocal shaking in a 500 ml Sakaguchi flask for 20hours at 30° C. Cell pellets were recovered by centrifuging (3,000×g, 10min.) 10 L of the resulting culture. The resulting cell pellets werewashed twice with 1 L of 0.1M potassium phosphate buffer (pH7.0) toprovide 119 g of the cell pellets.

These cell pellets were suspended in 0.1M potassium phosphate buffer(pH7.0) at 2 ml for 1 g of the cell pellets. A millimolar of (1 mM)PMSF, 5 mM of benzamidine HCl, 1 mM of EDTA, 3 μg/ml of leupeptin, 3μg/ml of pepstatin and 1 mM of dithiotritol were added. A cell lysatesolution was obtained by disrupting twice repetitively the suspensionwith a French press (1000 kg/cm²) (Ohtake Seisakusho). Aftercentrifuging the cell lysate solution (40,000×g, 30 minutes), thesupernatant was recovered and centrifuged for 1 hour at 150,000×g torecover the supernatant (hereinafter referred to as the “crude cellextract”).

(2) Determination of the Ability of Converting Compound (II) to Compound(III)

There was prepared 30 μl of a reaction solution of 0.1M potassiumphosphate buffer (pH7.0) containing 3 ppm of compound (II) labeled with¹⁴C, 2.4 mM of β-NADPH (hereinafter, referred to as “component A”)(Oriental Yeast Company), 0.5 mg/ml of a ferredoxin derived from spinach(hereinafter referred to as “component B”) (Sigma Company), 1 U/ml offerredoxin reductase (hereinafter, referred to as “component C”) (SigmaCompany) and 18 μl of the crude cell extract recovered in Example 5(1).The reaction solution was maintained at 30° C. for a hour. Further,there was prepared and maintained similarly a reaction solution havingno addition of at least one component utilized in the composition of theabove reaction solution, selected from component A, component B andcomponent C. Three microliters (3 μl) of 2N HCl and 90 μl of ethylacetate were added and mixed into each of the reaction solutions afterthe maintenance. The resulting reaction solutions were centrifuged at8,000×g to recover 75 μl of the ethyl acetate layer. After drying theethyl acetate layers under reduced pressure, the residue was dissolvedin 6.0 μl of ethyl acetate. Five microliters (5.0 μl) thereof wasspotted to a TLC plate (TLC plate silica gel 60F₂₅₄ 20 cm×20 cm, 0.25thick, Merck Company). The TLC plate was developed with a 6:1:2 mixtureof chloroform, acetic acid and ethyl acetate for about 1 hour. Thesolvents were then allowed to evaporate. The TLC plate was exposedovernight to an imaging plate (Fuji Film Company). Next, the imagingplate was analyzed on Image Analyzer BAS2000 (Fuji Film Company). Thepresence of a spot corresponding to compound (III) labeled with ¹⁴C wereexamined (Rf value 0.24 and 0.29). The results are shown in Table 7.TABLE 7 Reaction components component component component crude cellcompound (II) spot of A B C extract labeled with¹⁴C compound (III) + + +− + − + + + + + + − + + + + − + − − + + −(3) Fractionation of the Crude Cell Extract

Ammonium sulfate was added to the crude cell extract obtained in Example5(1) to amount to 45% saturation. After stirring in ice-cooledconditions, the supernatant was recovered by centrifuging for 10 minutesat 12,000×g. After adding ammonium sulfate to the obtained supernatantto amount to 55% saturation and stirring in ice-cooled conditions, apellet was recovered by centrifuging for 10 minutes at 12,000×g. Thepellet was dissolved with 32.5 ml of 20 mM bistrispropane buffer(pH7.0). This solution was subjected to a PD10 column (AmershamPharmacia Company) and eluted with 20 mM of bistrispropane buffer(pH7.0) to recover 45.5 ml of fractions containing proteins (hereinafterreferred to as the “45-55% ammonium sulfate fraction”).

(4) Isolation of the Present Invention Protein (A2)

The 45-55% ammonium sulfate fraction prepared in Example 5(3) wasinjected into a HiLoad26/10 Q Sepharose HP column (Amersham PharmaciaCompany). Next, after flowing 100 ml of 20 mM bistrispropane buffer(pH7.0) into the column, 20 mM bistrispropane buffer was flown with alinear gradient of NaCl (gradient of NaCl was 0.004M/minute, range ofNaCl concentration was from 0M to 0.5M, flow rate was 8 ml/minute) tofraction recover 30 ml of fractions eluting at the NaCl concentration offrom 0.25M to 0.26M. Further, the recovered fractions were subjected toa PD10 column (Amersham Pharmacia Biotech Company) and eluted with 2 mMbistrispropane buffer (pH7.0) to recover the fractions containingprotein.

The recovered fractions were subjected to a PD10 column (AmershamPharmacia Biotech Company) with the elution with Buffer A (2 mMpotassium phosphate buffer containing 1.5 mM of NaCl, pH 7.0), in orderto recover the fractions containing protein. Next, the fractions wereinjected into a Bio-Scale Ceramic Hydroxyapatite Type I column CHT10-I(BioRad Company). Twenty milliliters (20 ml) of Buffer A was flown intothe column. Subsequently, Buffer A was flown with a linear gradient ofBuffer B (100 mM potassium phosphate buffer containing 0.03 mM of NaCl;the linear gradient started at 100% Buffer A to increase to 50% Buffer Bover a 100 minute period, flow rate was 2 ml/minute) to fraction recover10 ml of fractions eluting at a Buffer B concentration of from 23% to25%. Further, the recovered fractions were subjected to a PD10 column(Amersham Pharmacia Biotech Company) and eluted with 0.05M potassiumphosphate buffer (pH7.0) to recover the fractions containing protein.

The recovered fractions were concentrated to about 770 μl using anultrafilter membrane (Microcon YM-30, Millipore Company) and injectedinto a HiLoad 16/60 Superdex 75 pg column (Amersham Pharmacia BiotechCompany). Fifty millimolar (50 mM) potassium phosphate buffer containing0.15M of NaCl (pH7.0) was flown (flow rate 1 ml/minute) into the column,The elution was fractioned at 2 ml each. The fractions eluting at theelution volumes of more or less 61 ml were each fraction recovered. Theprotein contained in each of the fractions was analyzed with a 10%-20%SDS-PAGE.

Instead of the crude cell extract in the reaction solution described inExample 5(2), the recovered fractions were added and maintained in thepresence of component A, component B, component C and compound (II)labeled with ¹⁴C, similarly to Example 5(2). The reaction solutionsafter the maintenance were TLC analyzed to examine the intensity of thespots corresponding to compound (III) labeled with ¹⁴C. The proteinmoving to the position to 47 kDa in the above SDS-PAGE was observed tohave its fluctuations in the concentrations of the bands of thefractions added in turn to be parallel with the fluctuations of theintensity of the spots corresponding to compound (III). Said protein wasrecovered from the SDS-PAGE gel and was subjected to an amino acidsequence analysis with a protein sequencer (Applied Biosystems Company,Procise 494HT, pulsed liquid method) to sequence the N terminus aminoacid sequence. As a result, the amino acid sequence shown in SEQ ID NO:20 was provided. Further, after digesting the above protein withtrypsin, the obtained digestion material was analyzed on a massspectrometer (ThermoQuest Company, Ion Trap Mass Spectrometer LCQ,column: LC Packings Company PepMap C18 75 μm×150 mm, solvent A: 0.1%HOAc—H₂O, solvent B: 0.1% HOAc-methanol, gradient: a linear gradientstarting at an elution with a mixture of 95% of solvent A and 5% ofsolvent B and increasing to a concentration of 100% of solvent B over 30minutes, flow rate: 0.2 μl/minute). As a t result, the sequence shown inSEQ ID NO: 21 was provided.

Example 6 Obtaining the Present Invention DNA (A2)

(1) Preparation of the Chromosomal DNA of Saccharopolyspora taberi JCM9383t

Saccharopolyspora taberi JCM 9383t was shake cultured at 30° C. for 1day to 3 days in 50 ml of YEME medium (0.3%(w/v) yeast extract,0.5%(w/v) bacto-peptone, 0.3%(w/v) malt extract, 1.0%(w/v) glucose,34%(w/v) sucrose and 0.2%(v/v) 2.5M MgCl₂.6H₂O). The cells wererecovered. The obtained cells were suspended in YEME medium containing1.4%(w/v) glycine and 60 mM EDTA and further incubated with shaking fora day. The cells were recovered from the culture medium. After washingonce with distilled water, it was resuspended in buffer (100 mM Tris-HCl(pH8.0), 100 mM EDTA, 10 mM NaCl) at 1 ml per 200 mg of the cellpellets. Two hundred micrograms per milliliter (200 μg/ml) of egg-whitelysozyme were added. The cell suspension was shaken at 30° C. for ahour. Further, 0.5% of SDS and 1 mg/ml of Proteinase K was added. Thecell suspension was incubated at 55° C. for 3 hours. The cell suspensionwas extracted twice with phenol.chloroform.isoamyl alcohol to recovereach of the aqueous layers. Next, there was one extraction withchloroform.isoamyl alcohol to recover the aqueous layer. The chromosomalDNA was obtained by ethanol precipitating the aqueous layer.

(2) Preparation of the Chromosomal DNA Library of Saccharopolysporataberi JCM 9383t

Nineteen micrograms (19 μg) of the chromosomal DNA prepared in Example5(1) were digested with 0.78 U of restriction enzyme Sau3AI at 37° C.for 60 minutes. The obtained digestion solution was separated with 1%agarose gel electrophoresis. The DNA of about 2.0 kbp was recovered fromthe gel. The DNA was purified with QIAquick Gel Extraction Kit (QiagenCompany) according to the instructions attached to said kit and wasconcentrated with an ethanol precipitation to obtain 10 μl of thesolution containing the target DNA. Eight microliters (8 μl) of the DNAsolution, 100 ng of plasmid vector pUC118 digested with restrictionenzyme BamHI and treated with dephosphorylation and 12 μli of the Isolution from Ligation Kit Ver. 2 (Takara Shuzo Company) were mixed andmaintained for 3 hours at 16° C. E coli DH5α was transformed with theligation solution. The E. coli transformants were cultured overnight at37° C. in LB agar medium containing 50 mg/l of ampicillin. The obtainedcolonies were recovered from an agar medium. The plasmids were extractedand were designated as the chromosomal DNA library.

(3) Isolation of the Present Invention DNA (A2)

PCR was conducted by utilizing the chromosomal DNA prepared in Example6(1) as the template with Expand HiFi PCR System (Boehringer ManheimCompany) (FIG. 2). As the primers, there was utilized the pairing of anoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 54and an oligonucleotide having the nucleotide sequence shown in SEQ IDNO: 55 (hereinafter referred to as “primer paring 6”). The nucleotidesequence shown in SEQ ID NO: 54 was designed based on a nucleotidesequence encoding the N terminus amino acid sequence shown in SEQ ID NO:20. Further, the nucleotide sequence shown in SEQ ID NO: 55 was designedbased on a nucleotide sequence complimentary to the nucleotide sequenceencoding the inner amino acid sequence shown in SEQ ID NO: 21. The PCRreaction solution amounted to 25 μl by adding 300 ng of the abovechromosomal DNA, the 2 primers each amounting to 7.5 pmol, 0.2 μl ofdNTP mix (a mixture of 2 mM of each of the 4 types of dNTP), 2.5 μl of10× buffer (containing MgCl₂), 0.19 μl of Expand HiFi enzyme mix anddistilled water. The reaction conditions of the PCR were aftermaintaining 97° C. for 2 minutes, repeating 10 cycles of a cycle thatincluded maintaining 97° C. for 15 seconds, followed by 65° C. for 30seconds and followed by 72° C. for 1 minute; then conducting 15 cyclesof a cycle that included maintaining 97° C. for 15 seconds, followed by65° C. for 30 seconds and followed by 72° C. for 1 minute (wherein 20seconds was added to the maintenance at 72° C. for each cycle); and thenmaintaining 72° C. for 7 minutes. After the maintenance, the reactionsolution was subjected to 2% agarose gel electrophoresis. The gel areacontaining the DNA of about 800 bp was recovered. The DNA was purifiedfrom the recovered gel by utilizing Qiagen quick gel extraction kit(Qiagen Company) according to the attached instructions. The obtainedDNA was ligated to the TA cloning vector pCRII-TOPO (Invitrogen Company)according to the instructions attached to said vector and was introducedinto E. Coli TOP10F′. The plasmid DNA was prepared from the obtained E.coli transformant, utilizing Qiagen Tip20 (Qiagen Company). A sequencingreaction was conducted with Dye terminator cycle sequencing FS readyreaction kit (Applied Biosystems Japan Company) according to theinstructions attached to said kit, utilizing as primers the −21M13primer (Applied Biosystems Japan Company) and M13Rev primer (AppliedBiosystems Japan Company). The reaction products were analyzed with aDNA sequencer 373A (Applied Biosystems Japan Company). As a result, thenucleotide sequence shown in nucleotides 36 to 819 of the nucleotidesequence shown in SEQ ID NO: 10 was provided. Nucleotides 37-60 of thenucleotide sequence shown in SEQ ID NO: 10 encoded a part of the aminoacid sequence shown in SEQ ID NO: 20. In this regard, it was expectedthat that said DNA encoded a part of the present invention protein (A2).

Next, PCR was conducted by utilizing the chromosomal DNA prepared inExample 6(2) as the template and similar to the above with Expand HiFiPCR system. There was utilized as primers, a pairing of anoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 56with an oligonucleotide having the nucleotide sequence shown in SEQ IDNO: 57 (hereinafter referred to as the “primer pairing 7”). Byconducting the PCR with such primers, there was amplified a DNA having anucleotide sequence in which the 5′ terminus elongates past thenucleotide shown as nucleotide 36 of the nucleotide sequence shown inSEQ ID NO: 10. Further, there was utilized as primers, a pairing of anoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 58with an oligonucleotide having the nucleotide sequence shown in SEQ IDNO: 59 (hereinafter referred to as the “primer pairing 8”). Byconducting the PCR with such primers, there was amplified a DNA having anucleotide sequence in which the 3′ terminus elongates past thenucleotide shown as nucleotide 819 of the nucleotide sequence shown inSEQ ID NO: 10. Each of the 1.3 kb DNA amplified with the use of primerpairing 7 and the 0.4 kb DNA amplified with the use of primer pairing 8was cloned into TA cloning vector pCRII-TOPO. Plasmid DNA was preparedfrom the obtained E. coli transformant, utilizing Qiagen Tip 20 (QiagenCompany). A sequencing reaction was conducted with Dye terminator cyclesequencing FS ready reaction kit (Applied Biosystems Japan Company)according to the instructions attached to said kit, utilizing as primersthe −21M13 primer (Applied Biosystems Japan Company), M13Rev primer(Applied Biosystems Japan Company) and the oligonucleotide shown in SEQID NO: 60. The reaction products were analyzed with a DNA sequencer 373A(Applied Biosystems Japan Company). As a result of sequencing thenucleotide sequence of the 1.3 kb DNA amplified by utilizing primerpairing 7, the nucleotide sequence shown in nucleotides 1 to 35 of thenucleotide sequence shown in SEQ ID NO: 10 was provided. Further, as aresult of sequencing the nucleotide sequence of the 0.4 kb DNA amplifiedby utilizing primer pairing 8, the nucleotide sequence shown innucleotides 819 to 1415 of the nucleotide sequence shown in SEQ ID NO:10 was provided. As a result of connecting the obtained nucleotidesequences, the nucleotide sequence shown in SEQ ID NO: 10 was obtained.Two open reading frames (ORF) were present in said nucleotide sequence.As such, there was contained a nucleotide sequence (SEQ ID NO: 7)consisting of 1206 nucleotides (inclusive of the stop codon) andencoding a 401 amino acid residue as well as a nucleotide sequence (SEQID NO: 16) consisting of 198 nucleotides (inclusive of te stop codon)and encoding a 65 amino acid residue. The molecular weight of theprotein consisting of the amino acid sequence (SEQ ID NO: 2) encoded bythe nucleotide sequence shown in SEQ ID NO: 7 was calculated to be 43983Da Further, the amino acid sequence encoded by said nucleotide sequencecontained the amino acid sequence (SEQ ID NO: 20) determined from theamino acid sequencing of from the N terminus of the present inventionprotein (A2) and the amino acid sequence (SEQ ID NO: 21) determined fromthe amino acid sequencing of the mass spectrometer analysis with thetrypsin digestion fragments. The molecular weight of the proteinconsisting of the amino acid sequence (SEQ ID NO: 13) encoded by thenucleotide sequence shown in SEQ ID NO: 16 was calculated be 6707 Da.

Example 7 Expression of the Present Invention Protein (A2) in E. coli

(1) Production of a Transformed E. coli Having the Present InventionProtein (A2)

PCR was conducted by utilizing as a template the chromosomal DNAprepared from Saccharopolyspora taberi JCM 9383t in Example 6(1) and byutilizing Expand HiFi PCR System (Boehringer Manheim Company). As theprimers, there was utilized the pairing of an oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 61 and an oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 62 (hereinafter referred toas “primer pairing 21”) or a pairing of an oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 61 and an oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 63 (hereinafter referred toas “primer pairing 22”). The PCR reaction solution amounted to 50 μl byadding the 2 primers each amounting to 300 nM, 50 ng of the abovechromosomal DNA, 5.0 μl of dNTP mix (a mixture of 2.0 mM of each of the4 types of dNTP), 5.0 μl of 10× Expand HF buffer (containing MgCl₂) and0.75 μl of Expand HiFi enzyme mix and distilled water. The reactionconditions of the PCR were after maintaining 97° C. for 2 minutes;repeating 10 cycles of a cycle that included maintaining 97° C. for 15seconds, followed by 60° C. for 30 seconds and followed by 72° C. for 1minute; then conducting 15 cycles of a cycle that included maintaining97° C. for 15 seconds, followed by 60° C. for 30 seconds and followed by72° C. for 1 minute (wherein 20 seconds was added to the maintenance at72° C. for each cycle); and then maintaining 72° C. for 7 minutes. Afterthe maintenance, the reaction solution was subjected to 1% agarose gelelectrophoresis. The gel area containing the DNA of about 1.2 kbp wasrecovered from the gel which was subjected the reaction solutionutilizing primer pairing 21. The gel area containing the DNA of about1.4 kbp was recovered from the gel which was subjected the reactionsolution utilizing primer pairing 22. The DNA were purified from each ofthe recovered gels by utilizing Qiagen quick gel extraction kit (QiagenCompany) according to the attached instructions. The obtained DNA wereligated to the cloning vector pCRII-TOPO (Invitrogen Company) accordingto the instructions attached to said vector and were introduced into E.Coli TOP10F′. The plasmid DNA were prepared from the obtained E. colitransformants, utilizing Qiagen Tip20 (Qiagen Company). Next, sequencingreactions were conducted with Dye terminator cycle sequencing FS readyreaction kit (Applied Biosystems Japan Company) according to theinstructions attached to said kit, utilizing as primers the −21M13primer (Applied Biosystems Japan Company), M13Rev primer (AppliedBiosystems Japan Company), the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 56 and the oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 64. The reaction products wereanalyzed with a DNA sequencer 373A (Applied Biosystems Japan Company).Based on the results, the plasmid having the nucleotide sequence shownin SEQ ID NO: 7 was designated as pCR923 and the plasmid having thenucleotide sequence shown in SEQ ID NO: 10 was designated as pCR923F.

Next, each of plasmids pCR923 and pCR923F was digested with restrictionenzymes NdeI and HindIII. The digestion products were subjected toagarose gel electrophoresis The gel area containing a DNA of about 1.2kbp was cut from the gel subjected to the digestion products of pCR923.The gel area containing a DNA of about 1.4 kbp was cut from the gelsubjected to the digestion products of pCR923F. The DNA were purifiedfrom each of the recovered gels by utilizing Qiagen quick gel extractionkit (Qiagen Company) according to the attached instructions. Each of theobtained DNA and the plasmid pKSN2 digested with NdeI and HindIII wereligated with ligation kit Ver.1 (Takara Shuzo Company) according to theinstructions attached to said kit and introduced into E. Coli JM109. Theplasmid DNA were prepared from the obtained E. coli transformants. Thestructures thereof were analyzed. The plasmid containing the nucleotidesequence shown in SEQ ID NO: 7, in which the DNA of about 1.2 kbpencoding the present invention protein (A2) is inserted between the NdeIsite and the HindIII site of pKSN2 was designated as pKSN923 further,the plasmid containing the nucleotide sequence shown in SEQ ID NO: 10,in which the DNA of about 1.4 kbp encoding the present invention protein(A2) is inserted between the NdeI site and the HindIII site of pKSN2 wasdesignated as pKSN923F. Each of the above plasmids of pKSN923 andpKSN923F was introduced into E. coli JM109. The obtained E. colitransformants were designated, respectively, JM109/pKSN923 andM109/pKSN923F. Further, plasmid pKSN2 was introduced into E. coli JM109.The obtained E. coli transformant was designated as JM109/pKSN2.

(2) Expression of the Present Invention Protein (A2) in E. coli andRecovery of Said Protein

E. coli JM109/pKSN657, JM109/pKSN657F and JM109/pKSN2 were each culturedovernight at 37° C. in 10 ml of TB medium (1.2%(w/v) tryptone, 2.4%(w/v)yeast extract, 0.4%(w/v) glycerol, 17 mM potassium dihydrogenphosphate,72 mM dipotassium hydrogenphosphate) containing 50 μm/ml of ampicillin.A milliliter (1 ml) of the obtained culture medium was transferred to100 ml of TB medium containing 50 μg/ml of ampicillin and cultured at26° C. When OD660 reached about 0.5, 5-aminolevulinic acid was added tothe final concentration of 500 μM, and the culturing was continued.Thrity (30) minutes thereafter, IPTG was added to a final concentrationof 1 mM, and there was further culturing for 17 hours.

The cells were recovered from each of the culture mediums, washed with0.1M tris-HCl buffer (pH7.5) and suspended in 10 ml of said buffercontaining 1 mM PMSF. The obtained cell suspensions were subjected 6times to a sonicator (Sonifier (Branson Sonic Power Company)) at 3minutes each under the conditions of output 3, duty cycle 30%, in orderto obtain cell lysate solutions. After centrifuging the cell lysatesolutions (1,200×g, 5 minutes) the supernatants were recovered andcentrifuged (150,000×g, 70 minutes) to recover supernatant fractions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN923 is referred to as “E. coli pKSN923 extract”, thesupernatant fraction obtained from E. coli JM109/pKSN923F is referred toas “E. coli pKSN923F extract”, and the supernatant fraction obtainedfrom E. coli JM109/pKSN2 is referred to as “E. coli pKSN2 extract”). Amicroliter (1 μl) of the above supernatant fractions was analyzed on a15% to 25% SDS-PAGE and stained with CBB. As a result, notably moreintense bands were detected in both E. coli pK SN923 extract and E. colipKSN923F extract than the E. coli pKSN2 extract, at the electrophoresislocations corresponding to the molecular weight of 47 kDa. It wasconfirmed that E. coli JM109/pKSN923 and E. coli JM109/pKSN923Fexpressed the present invention protein (A2).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Reaction solutions of 30 μl were prepared and maintained for 10 minutesat 30° C. The reaction solutions consisted of a 0.1M potassium phosphatebuffer (pH7.0) containing 3 ppm of compound (II) labeled with ¹⁴C, 2 mMof β-NADPH (hereinafter, referred to as “component A”) (Oriental YeastCompany), 0.2 mg/ml of a ferredoxin derived from spinach (hereinafterreferred to as “component B”) (Sigma Company), 1 U/ml of ferredoxinreductase (hereinafter, referred to as “component C”) (Sigma Company)and 18 μl of the supernatant fraction recovered in Example 7(2).Further, there were prepared and maintained similarly reaction solutionshaving no addition of at least one component utilized in the compositionof the above reaction solution, selected from component A, component Band component C. Three microliters (3 μl) of 2N HCl and 90 μl of ethylacetate were added and mixed into each of the reaction solutions afterthe maintenance. The resulting reaction solutions were centrifuged at8,000×g to recover 75 μl of the ethyl acetate layer. After drying theethyl acetate layers under reduced pressure, the residue was dissolvedin 6.0 μl of ethyl acetate. Five microliters (5.0 μl) thereof wasspotted to a silica gel TLC plate (TLC plate silica gel 60F₂₅₄, 20 cm×20cm, 0.25 mm thick, Merck Company). The TLC plate was developed with a6:1:2 mixture of chloroform, acetic acid and ethyl acetate for about 1hour. The solvents were then allowed to evaporate. The TLC plate wasexposed overnight to an imaging plate (Fuji Film Company). Next, theimaging plate was analyzed on Image Analyzer BAS2000 (Fuji FilmCompany). The presence of a spot corresponding to compound (III) labeledwith ¹⁴C were examined (of value 0.24 and 0.29). The results are shownin Table 8. TABLE 8 Reaction components component component component E.coli compound (II) spot of A B C extract labeled with¹⁴C compound(III) + + + − + − + + + pKSN2 + − + + + pKSN923 + + − + + pKSN923 + − +− + pKSN923 + − + + − pKSN923 + + + + + pKSN923F + + − + + pKSN923F +− + − + pKSN923F + − + + − pKSN923F + +

Example 8 Preparation of the Present Protein (A10)

(1) Preparation of the Crude Cell Extract

A frozen stock of Streptomyces griseolus ATCC 11796 was added to 250 mlof B medium (1%(w/v) glucose, 0.1%(w/v) meat extract, 0.2%(w/v)tryptose) in a 500 ml baffled flask and incubated with rotary shaking at30° C. for 3 days to obtain a pre-culture. Forty milliliters (40 ml) ofthe pre-culture was added to 400 ml of B medium and was incubated withrotary shaking in a 1 L triangular flask at 30° C. for 24 hours. Afterstopping the culturing, the culture was allowed to settle. Two hundredand twenty milliliters (220 ml) of only the supernatant was removed. Twohundred and twenty milliliters (220 ml) of fresh medium similarlyprepared was added to the remaining 220 ml of the culture medium toamount to 440 ml. Compound (II) was added thereto to amount to 100 ppm.The cells were incubated with rotary shaking in the 1 L triangular flaskat 30° C. for 40 hours. Cell pellets were recovered by centrifuging(3,000 g, 5 min.) 2.6 L of the resulting culture. The resulting cellpellets were washed with 1 L of 0.1M PIPES-NaOH buffer (pH6.8) toprovide 26 g of the cell pellets.

These cell pellets were suspended of 0.1M PIPES-NaOH buffer (pH6.8) at 3ml for 1 g of the cell pellets, and 1 mM of PMSF, 5 mM of benzamidineHCl, 1 mM of EDTA, 3 μg/ml of leupeptin, 3 μg/ml of pepstatin A and 1 mMof dithiotritol were added. A cell lysate solution was obtained bydisrupting twice repetitively the suspension with a French press (1000kg/cm²) (Ohtake Seisakusho). After centrifuging the cell lysate solution(40,000×g, 30 minutes), the supernatant was recovered and centrifugedfor 1 hour at 150,00×g to recover the supernatant (hereinafter referredto as the “crude cell extract”).

(2) Determination of the Ability of Converting Compound (II) to Compound(III)

There was prepared 30 μl of a reaction solution of 0.1M potassiumphosphate buffer (pH7.0) containing 3 ppm of compound (II) labeled with¹⁴C, 2.4 mM of β-NADPH (hereinafter, referred to as “component A”)(Oriental Yeast Company), 0.5 mg/ml of a ferredoxin derived from spinach(hereinafter referred to as “component B”) (Sigma Company), 1 U/ml offerredoxin reductase (hereinafter, referred to as “component C”) (SigmaCompany) and 18 μl of the crude cell extract recovered in Example 8(1).The reaction solution was maintained at 30° C. for a hour. Further,there was prepared and maintained similarly a reaction solution havingno addition of at least one component utilized in the composition of theabove reaction solution, selected from component A, component B andcomponent C. Three microliters (3 μl) of 2N HCl and 90 μl of ethylacetate were added and stirred into each of the reaction solutions afterthe maintenance. The resulting reaction solutions were centrifuged at8,000×g to recover 75 μl of the ethyl acetate layer. After drying theethyl acetate layers under reduced pressure, the residue was dissolvedin 6.0 μl of ethyl acetate. Five microliters (5.0 μl) thereof wasspotted to a silica gel TLC plate (TLC plate silica gel 60F₂₅₄, 20 cm×20cm, 0.25 thick, Merck Company). The TLC plate was developed with a 6:1:2mixture of chloroform, acetic acid and ethyl acetate for about 1 hour.The solvents were then allowed to evaporate. The TLC plate was exposedovernight to an imaging plate (Fuji Film Company). Next, the imagingplate was analyzed on Image Analyzer BAS2000 (Fuji Film Company). Thepresence of a spot corresponding to compound (III) labeled with ¹⁴C wereexamined (Rf value 0.24 and 0.29). The results are shown in Table 9.TABLE 9 Reaction components component component component crude cellcompound (II) spot of A B C extract labeled with¹⁴ C compound(III) + + + − + = + + + + + + − + + + + − + − − + + −(3) Fractionation of the Crude Cell Extract

Ammonium sulfate was added to the crude cell extract obtained in Example8(1) to amount to 45% saturation, After stirring in ice-cooledconditions, the supernatant was recovered by centrifuging for 10 minutesat 12,000×g. After adding ammonium sulfate to the obtained supernatantto amount to 55% saturation and stirring in ice-cooled conditions, apellet was recovered by centrifuging for 10 minutes at 12,000×g. Thepellet was dissolved with 20 mM bistrispropane buffer (pH7.0) to amountto 10 ml. This solution was subjected to a PD10 column (AmershamPharmacia Company) and eluted with 20 mM of bistrispropane buffer(pH7.0) to recover 14 ml of fractions containing proteins (hereinafterreferred to as the “45-55% ammonium sulfate fraction”).

(4) Isolation of the Present Protein (A10)

The 45-55% ammonium sulfate fraction prepared in Example 8(3) wasinjected into a MonoQ HR 10/10 column (Amersham Pharmacia Company).Next, after flowing 16 ml of 20 mM bistrispropane buffer (pH7.0) intothe column, 20 mM bistrispropane buffer was flown with a linear gradientof NaCl (gradient of NaCl was 0.00625M/minute, range of NaClconcentration was from 0M to 0.5M, flow rate was 4 ml/minute) tofraction recover 15 ml of fractions eluting at the NaCl concentration offrom 0.28M to 0.31M. Further, the recovered fractions were subjected toa PD10 column (Amersham Pharmacia Biotech Company) and eluted with 20 mMbistrispropane buffer (pH7.0) to recover the fractions containingprotein.

The recovered fractions were subjected to a PD10 column (AmershamPharmacia Biotech Company) with the elution with Buffer A (2 mMpotassium phosphate buffer containing 1.5 mM of NaCl, pH 7.0), in orderto recover the fractions containing protein. Next, the fractions wereinjected into a Bio-Scale Ceramic Hydroxyapatite Type I column CHT10-I(BioRad Company). Fifty milliliters (50 ml) of Buffer A was flown intothe column. Subsequently, Buffer A was flown with a linear gradient ofBuffer B (100 mM potassium phosphate buffer containing 0.03 mM of NaCl;the linear gradient started at 100% Buffer A to increase to 50% Buffer Bover a 40 minute period, flow rate was 5 ml/minute) to fraction recoverthe fractions eluting at a Buffer B concentration of from 16% to 31%.Further, the recovered fractions were subjected to a PD10 column(Amersham Pharmacia Biotech Company) and eluted with 0.05M potassiumphosphate buffer (pH7.0) to recover the fractions containing protein.The protein contained in each of the fractions were analyzed on a10%-20% SDS-PAGE.

Instead of the crude cell extract in the reaction solution described inExample 8(2), the recovered fractions were added and maintained in thepresence of component A, component B, component C and compound (II)labeled with ¹⁴C, similarly to Example 8(2). The reaction solutionsafter the maintenance were TLC analyzed to examine the intensity of thespots corresponding to compound (III) labeled with ¹⁴C. The proteinmoving to the position to 47 kDa in the above SDS-PAGE was observed tohave its fluctuations in the concentrations of the bands of thefractions added in turn to be parallel with the fluctuations of theintensity of the spots corresponding to compound (III). Said protein wasrecovered from the SDS-PAGE gel and digested with trypsin. The obtaineddigestion material was analyzed on a mass spectrometer (ThermoQuestCompany, Ion Trap Mass Spectrometer LCQ, column: LC Packings CompanyPepMap C18 75 μm×150 mm, solvent A: 0.1% HOAc—H₂O, solvent B: 0.1%HOAc-methanol, gradient: a linear gradient starting at an elution with amixture of 95% of solvent A and 5% of solvent B and increasing to aconcentration of 100% of solvent B over 30 minutes, flow rate: 0.2μl/minute). As a result, the amino acid sequences shown in each and anyone of SEQ ID NO: 22-34 were provided.

Example 9 Preparation of the Chromosomal DNA of Streptomyces GriseolusATCC 11796

Streptomyces griseolus ATCC 11796 was incubated with shaking at 30° C.for 1 day to 3 days in 50 ml of YEME medium (0.3%(w/v) yeast extract,0.5%(w/v) bacto-peptone, 0.3%(w/v) malt extract, 1.0%(w/v) glucose,34%(w/v) sucrose and 0.2%(v/v) 2.5M MgCl₂ 6H₂O). The cells wererecovered. The obtained cells were suspended in YEME medium containing1.4%(w/v) glycine and 60 mM EDTA and further incubated with shaking fora day. The cells were recovered from the culture medium. After washingonce with distilled water, it was resuspended in buffer (100 mM Tris-HCl(pH8.0), 100 mM EDTA, 10 mM NaCl) at 1 ml per 200 mg of the cells. Twohundred micrograms per milliliter (200 μg/ml) of egg-white lysozyme wereadded. The cell suspension was shaken at 30° C. for a hour. Further,0.5% of SDS and 1 mg/ml of Proteinase K was added. The cell suspensionwas incubated at 55° C. for 3 hours. The cell suspension was extractedtwice with phenol.chloroform.isoamyl alcohol to recover each of theaqueous layers. Next, there was one extraction with chloroform.isoamylalcohol to recover the aqueous layer. The chromosomal DNA was obtainedby ethanol precipitating the aqueous layer.

Example 10 Obtaining a DNA Encoding the Present DNA (A10) and Expressionin E. coli

(1) Production of a Transformed E. coli Having the Present DNA

PCR was conducted by utilizing as a template the chromosomal DNAprepared from Streptomyces griseolus ATCC 11796 in Example 9 and byutilizing Expand High Fidelity PCR System (Roche Molecular BiochemicalsCompany). As the primers, there was utilized the pairing of anoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 79and an oligonucleotide having the nucleotide sequence shown in SEQ IDNO: 80 (hereinafter referred to as “primer pairing 23”) or a pairing ofan oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 79and an oligonucleotide having the nucleotide sequence shown in SEQ IDNO: 81 (hereinafter referred to as “primer pairing 24”). The PCRreaction solutions amounted to 501 μl by adding the 2 primers eachamounting to 300 nM, 50 ng of the above chromosomal DNA, 5.0 μl of dNTPmix (a mixture of 2.0 mM of each of the 4 types of dNTP), 5.0 μl of 10×Expand HF buffer (containing MgCl₂) and 0.75 μl of Expand HiFi enzymemix and distilled water. The reaction conditions of the PCR were aftermaintaining 97° C. for 2 minutes; repeating 10 cycles of a cycle thatincluded maintaining 97° C. for 15 seconds, followed by 65° C. for 30seconds and followed by 72° C. for 2 minutes; then conducting 15 cyclesof a cycle that included maintaining 97° C. for 15 seconds, followed by68° C. for 30 seconds and followed by 72° C. for 2 minutes (wherein 20seconds was added to the maintenance at 72° C. for each cycle); and thenmaintaining 72° C. for 7 minutes. After the maintenance, each of thereaction solutions was subjected to 1% agarose gel electrophoresis. Thegel area containing the DNA of about 1.2 kbp was recovered from the gelwhich was subjected the reaction solution utilizing primer pairing 23.The gel area containing the DNA of about 1.5 kbp was recovered from thegel which was subjected the reaction solution utilizing primer pairing24. The DNA were purified from each of the recovered gels by utilizingQiagen quick gel extraction kit (Qiagen Company) according to theattached instructions. The obtained DNA were ligated to the cloningvector pCR2.1-TOPO (Invitrogen Company) according to the instructionsattached to said vector and were introduced into E. Coli TOP10F′. Theplasmid DNA were prepared from the obtained E. coli transformants,utilizing Qiaprep Spin Miniprep Kit (Qiagen Company). Next, sequencingreactions were conducted with Dye terminator cycle sequencing FS readyreaction kit (Applied Biosystems Japan Company) according to theinstructions attached to said kit, utilizing as primers the −21M13primer (Applied Biosystems Japan Company), M13Rev primer (AppliedBiosystems Japan Company), the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 82 and the oligonucleotide having thenucleotide sequence shown in SEQ ID NO; 83. The sequencing reactionsutilized the obtained plasmid DNA as the template. The reaction productswere analyzed with a DNA sequencer 373A (Applied Biosystems JapanCompany). Based on the results, the plasmid having the nucleotidesequence shown in SEQ ID NO: 84 was designated as pCR11796 and theplasmid having the nucleotide sequence shown in SEQ ID NO: 85 wasdesignated as pCR11796F. Two open reading frames (ORF) were present insaid nucleotide sequence shown in SEQ ID NO: 85. As such, there wascontained a nucleotide sequence (SEQ ID NO: 84) consisting of 1221nucleotides (inclusive of the stop codon) and encoding a 406 amino acidresidue (the amino acid sequence shown in SEQ ID NO: 5) and a nucleotidesequence consisting of 210 nucleotides (inclusive of the stop codon) andencoding a 69 amino acid residue.

Next, each of pCR11796 and pCR11796F was digested with restrictionenzymes NdeI and HindIII. The digestion products were subjected toagarose gel electrophoresis. The gel area containing a DNA of about 1.2kbp was cut from the gel subjected to the digestion products ofpCR11796. The gel area containing a DNA of about 1.5 kbp was cut fromthe gel subjected to the digestion products of pCR11796F. The DNA werepurified from each of the recovered gels by utilizing Qiagen quick gelextraction kit (Qiagen Company) according to the attached instructions.Each of the obtained DNA and the plasmid pKSN2 digested with NdeI andHindIII were ligated with ligation kit Ver.1 (Takara Shuzo Company)according to the instructions attached to said kit and introduced intoE. Coli JM109. The plasmid DNA were prepared from the obtained E. colitransformants. The structures thereof were analyzed. The plasmidcontaining the nucleotide sequence shown in SEQ ID NO: 84, in which theDNA of about 1.2 kbp encoding the present protein (A10) is insertedbetween the NdeI site and the HindIII site of pKSN2 was designated aspKSN11796. Further, the plasmid containing the nucleotide sequence shownin SEQ ID NO: 85, in which the DNA of about 1.5 kbp encoding the presentprotein (A10) is inserted between the NdeI site and the HindIII site ofpKSN2 was designated as pKSN11796F. Each of the above plasmids ofpKSN11796 and pKSN11796F was introduced into E. coli JM109. The obtainedE. coli transformants were designated, respectively, JM109/pKSN11796 andJM109/pKSN11796F. Further, plasmid pKSN2 was introduced into E. coliJM109. The obtained E. coli transformant was designated as JM109/pKSN2.

(2) Expression of the Present Protein (A10) in E. coli and Recovery ofSaid Protein

E. coli JM109/pKSN11796, JM109/pKSN11796F and JM109/pKSN2 were eachcultured overnight at 37° C. in 10 ml of TB medium (1.2%(w/v) tryptone,2.4%(w/v) yeast extract, 0.4%(w/v) glycerol, 17 mM potassiumdihydrogenphosphate, 72 mM dipotassium hydrogenphosphate) containing 50μg/ml of ampicillin. A milliliter (1 ml) of the obtained culture mediumwas transferred to 100 ml of TB medium containing 50 μg/ml of ampicillinand cultured at 26° C. When OD660 reached about 0.5, 5-aminolevulinicacid was added to the final concentration of 500 μM, and the culturingwas continued. Thirty (30) minutes thereafter, IPTG was added to a finalconcentration of 1 mM, and there was further culturing for 17 hours.

The cells were recovered from each of the culture mediums, washed with0.1M tris-HCl buffer (pH7.5) and suspended in 10 ml of the above buffercontaining 1 mM PMSF. The obtained cell suspensions were subjected 6times to a sonicator (Sonifier (Branson Sonic Power Company)) at 3minutes each under the conditions of output 3, duty cycle 30%, in orderto obtain cell lysate solutions. After centrifuging the cell lysatesolutions (1,200×g, 5 minutes) the supernatants were recovered andcentrifuged (150,000×g, 70 minutes) to recover supernatant fractions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN11796 is referred to as “E. coli pKSN11796 extract”, thesupernatant fraction obtained from E. coli JM109/pKSN11796F is referredto as “E. coli pKSN11796F extract”, and the supernatant fractionobtained from E. coli JM109/pKSN2 is referred to as “E. coli pKSN2extract”) A microliter (1 μl) of the above supernatant fractions wasanalyzed on a 15% to 25% SDS-PAGE and stained with Coomasie Blue(hereinafter referred to as “CBB”). As a result, notably more intensebands were identified in both E. coli pKSN11796 extract and E. colipKSN11796F extract than the E. coli pKSN2 extract, at theelectrophoresis locations corresponding to the molecular weight of 45kDa. A more intense band was identified in E. coli pKSN11796F extractthan E. coli pKSN11796 extract. It was shown that E. coliJM109/pKSN11796F expressed the present protein (A10) to a higher degreethan E. coli JM109/pKSN11796.

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Reaction solutions of 30 μl were prepared and maintained for 1 hour at30° C. The reaction solutions consisted of a 0.1M potassium phosphatebuffer (pH7.0) containing 3 ppm of compound (II) labeled with ¹⁴C, 2 mMof β-NADPH (hereinafter, referred to as “component A”) (Oriental YeastCompany), 2 mg/ml of a ferredoxin derived from spinach (hereinafterreferred to as “component B”) (Sigma Company), 0.1 U/ml of ferredoxinreductase (hereinafter, referred to as “component C”) (Sigma Company)and 18 μl of the supernatant fraction recovered in Example 10(2).Further, there were prepared and maintained similarly reaction solutionshaving no addition of at least one component utilized in the compositionof the above reaction solution, selected from component A, component Band component C. Three microliters (3 μl) of 2N HCl and 90 μL of ethylacetate were added and mixed into each of the reaction solutions afterthe maintenance. The resulting reaction solutions were centrifuged at8,000×g to recover 75 μl of the ethyl acetate layer. After drying theethyl acetate layers under reduced pressure, the residue was dissolvedin 6.0 of ethyl acetate. Five microliters (5.0 μl) thereof was spottedto a silica gel TLC plate (TLC plate silica gel 60F₂₅₄ 20 cm×20 cm, 0.25mm thick, Merck Company). The TLC plate was developed with a 6:1:2mixture of chloroform, acetic acid and ethyl acetate for about 1 hour.The solvents were then allowed to evaporate. The TLC plate was exposedovernight to an imaging plate (Fuji Film Company). Next the imagingplate was analyzed on Image Analyzer BAS2000 (Fuji Film Company). Thepresence of a spot corresponding to compound (III) labeled with ¹⁴C wereexamined (Pf value 0.24 and 0.29). The results are shown in Table 10.TABLE 10 Reaction components component component component E. colicompound (II) spot of A B C extract labeled with¹⁴C compound (III) + + +− + − + + + pKSN2 + − + + + pKSN11796 + + − + + pKSN11796 + − + − +pKSN11796 + − + + − pKSN11796 + + + + + pKSN11796F + + − + +pKSN11796F + − + − + pKSN11796F + − + + − pKSN11796F + +

Example 11 Obtaining the Present Invention DNA (A3)

(1) Preparation of the Chromosomal DNA of Streptomyces testaceusATCC21469

Streptomyces testaceus ATCC21469 was incubated with shaking at 30° C.for 1 day to 3 days in 50 ml of YEME medium (0.3%(w/v) yeast extract,0.5%(w/v) bacto-peptone, 0.3%(w/v) malt extract, 1.0%(w/v) glucose,34%(w/v) sucrose and 0.2%(v/v) 2.5M MgCl₂.6H₂O). The cells wererecovered. The obtained cells were suspended in YEME medium containing1.4%(w/v) glycine and 60 mM EDTA and further incubated with shaking fora day. The cells were recovered from the culture medium. After washingonce with distilled water, it was resuspended in buffer (100 mM Tris-HCl(pH8.0), 100 mM EDTA, 10 mM NaCl) at 1 ml per 200 mg of the cells. Twohundred micrograms per milliliter (200 μg/1 ml) of egg-white lysozymewere added. The cell suspension was shaken at 30° C. for a hour.Further, 0.5% of SDS and 1 mg/ml of Proteinase K was added. The cellsuspension was incubated at 55° C. for 3 hours. The cell suspension wasextracted twice with phenol chloroform iso amyl alcohol to recover eachof the aqueous layers. Next, there was one extraction withchloroform.isoamyl alcohol to recover the aqueous layer. The chromosomalDNA was obtained by ethanol precipitating the aqueous layer.

(2) Isolation of the Present Invention DNA (A3)

PCR was conducted by utilizing the chromosomal DNA prepared in Example11(1) as the template. As the primers, there was utilized the pairing ofan oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 65and an oligonucleotide having the nucleotide sequence shown in SEQ IDNO: 66 (hereinafter referred to as “primer pairing 9”). The PCR reactionsolution amounted to 50 μl by adding 250 ng of the above chromosomalDNA, the 2 primers each amounting to 200 nM, 4 μl of dNTP mix (a mixtureof 2.5 mM of each of the 4 types of dNTP), 5 μl of 10× ExTaq buffer, 0.5μl of ExTaq polymerase (Takara Shuzo Company) and distilled water. Thereaction conditions of the PCR were maintaining 97° C. for 2 minutes;repeating 30 cycles of a cycle that included maintaining 97° C. for 15seconds, followed by 60° C. for 30 seconds and followed by 72° C. for 90seconds; and then maintaining 72° C. for 4 minutes. After themaintenance, the reaction solution was subjected to 0.8% agarose gelelectrophoresis. The gel area containing the DNA of about 1.4 kbp wasrecovered. The DNA was purified from the recovered gel by utilizingQIAquick gel extraction kit (Qiagen Company) according to the attachedinstructions. The obtained DNA was ligated to the TA cloning vectorpCR2.1 (Invitrogen Company) according to the instructions attached tosaid vector and was introduced into E. Coli TOP10F′. The plasmid DNA wasprepared from the obtained E. coli transformant, utilizing QIAprep SpinMiniprep Kit (Qiagen Company). A sequencing reaction was conducted withDye terminator cycle sequencing FS ready reaction kit (AppliedBiosystems Japan Company) according to the instructions attached to saidkit utilizing as primers the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 67 and the oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 68. The sequencing reactionsutilized the obtained plasmid as the template. The reaction productswere analyzed with a DNA sequencer 373A (Applied Biosystems JapanCompany). As a result, the nucleotide sequence shown in SEQ ID NO: 69was provided. Two open reading frames (ORF) were present in saidnucleotide sequence. As such, there was contained a nucleotide sequenceconsisting of 1188 nucleotides (inclusive of the stop codon) andencoding a 395 amino acid residue and a nucleotide sequence (SEQ ID NO:17) consisting of 195 nucleotides (inclusive of the stop codon) andencoding a 64 amino acid residue. The molecular weight of the amino acidsequence encoded by the nucleotide sequence shown in SEQ ID NO: 17 wascalculated to be 6666 Da.

Example 12 Expression of the Present Invention Protein (A3) in E. Coli

(1) Production of a Transformed E. coli Having the Present Invention DNA(A3)

PCR was conducted by utilizing as a template the chromosomal DNAprepared in Example 11(1) and by utilizing ExTaq polymerase (TakaraShuzo Company) under similar conditions as above. As the primers, therewas utilized the pairing of an oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 70 and an oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 71 (hereinafter referred to as“primer pairing 10”) or a pairing of an oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 70 and an oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 72 (hereinafter referred toas “primer pairing 11”). The DNA of 10.2 kb amplified by utilizing theprimer pairing 10 and the DNA of 1.5 kbp amplified by utilizing theprimer pairing 11 were cloned into TA cloning vector pCR2.1 according tothe above methods. The plasmid DNA were prepared from the obtained E.coli transformants, utilizing QIAprep Spin Miniprep Kit (QiagenCompany). Sequencing reactions were conducted with Dye terminator cyclesequencing FS ready reaction kit (Applied Biosystems Japan Company)according to the instructions attached to said kit utilizing as primersthe oligonucleotide having the nucleotide sequence shown in SEQ ID NO:67 and the oligonucleotide having the nucleotide sequence shown in SEQID NO: 68. The sequencing reactions utilized the obtained plasmid DNA asthe template. The reaction products were analyzed with a DNA sequencer373A (Applied Biosystems Japan Company). As a result, the plasmid clonedwith the DNA amplified by the primer pairing 10 was confirmed to havethe nucleotide sequence shown in SEQ ID NO: 8. The plasmid cloned withthe DNA amplified by primer pairing 11 was confirmed to have thenucleotide sequence shown in SEQ ID NO: 11. Two open reading frames(ORF) were present in said nucleotide sequence shown in SEQ ID NO: 11.As such, there was contained a nucleotide sequence (SEQ ID NO: 8)consisting of 1188 nucleotides (inclusive of the stop codon) andencoding a 395 amino acid residue and a nucleotide sequence consistingof 195 nucleotides (inclusive of the stop codon) and encoding a 64 aminoacid residue. The molecular weight of the protein consisting of theamino acid sequence encoded by the nucleotide sequence shown in SEQ IDNO: 8 was calculated to be 43752 Da. With the obtained plasmids, theplasmid having the nucleotide sequence shown in SEQ ID NO: 8 wasdesignated as pCR671 and the plasmid having the nucleotide sequenceshown in SEQ ID NO: 11 was designated as pCR671F.

Next, each of pCR671 and pCR671F was digested with restriction enzymesNdeI and HindIII. The digestion products were subjected to agarose gelelectrophoresis. The gel area containing a DNA of about 1.2 kbp was cutfrom the gel subjected to the digestion products of pCR671. The gel areacontaining a DNA of about 1.5 kbp was cut from the gel subjected to thedigestion products of pCR671F. The DNA were purified from each of therecovered gels by utilizing Qiagen quick gel extraction kit (QiagenCompany) according to the attached instructions. Each of the obtainedDNA and the plasmid pKSN2 digested with NdeI and HindIII were ligatedwith ligation kit Ver.1 (Takara Shuzo Company) according to theinstructions attached to said kit and introduced into E. Coli JM109. Theplasmid DNA were prepared from the obtained E. coli transformants. Thestructures thereof were analyzed. The plasmid containing the nucleotidesequence shown in SEQ ID NO: 8, in which the DNA of about 1200 bpencoding the present invention protein (A3) is inserted between the NdeIsite and the HindIII site of pKSN2 was designated as pKSN671. Further,the plasmid containing the nucleotide sequence shown in SEQ ID NO: 11,in which the DNA of about 1400 bp encoding the present invention protein(A3) is inserted between the NdeI site and the HindIII site of pKSN2 wasdesignated as pKSN671F. Each of the above plasmids of pKSN671 andpKSN671F was introduced into E. coli JM109. The obtained E. colitransformants were designated, respectively, JM109/pKSN671 andJM109/pKSN671F. Further, plasmid pKSN2 was introduced into E. coliJM109. The obtained E. coli transformant was designated as JM109/pKSN2.

(2) Expression of the Present Invention Protein (A3) in E. coli andRecovery of said Protein

E. coli JM109/pKSN671, JM109/pKSN671F and JM109/pKSN2 were each culturedovernight at 37° C. in 10 ml of TB medium (1.2%(w/v) tryptone, 2.4%(w/v)yeast extract, 0.4%(w/v) glycerol, 17 mM potassium dihydrogenphosphate,72 mM dipotassium hydrogenphosphate) containing 50 μg/ml of ampicillin.A milliliter (1 ml) of the obtained culture medium was transferred to100 ml of TB medium containing 50 μg/ml of ampicillin and cultured at26° C. When OD660 reached about 0.5, 5-aminolevulinic acid was added tothe final concentration of 500 μM, and the culturing was continued.Thirty (30) minutes thereafter, IPTG was added to a final concentrationof 1 mM, and there was further culturing for 17 hours.

The cells were recovered from each of the culture mediums, washed with0.1M tris-HCl buffer (pH7.5) and suspended in 10 ml of said buffercontaining 1 mM PMSF. The obtained cell suspensions were subjected 6times to a sonicator (Sonifier (Branson Sonic Power Company)) at 3minutes each under the conditions of output 3, duty cycle 30%, in orderto obtain cell lysate solutions. After centrifuging the cell lysatesolutions (1,200×g, 5 minutes) the supernatants were recovered andcentrifuged (150,000×g, 70 minutes) to recover supernatant fractions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN671 is referred to as “E. coli pKSN671 extract”, thesupernatant fraction obtained from E. coli JM109/pKSN671F is referred toas “E. coli pKSN671F extract”, and the supernatant fraction obtainedfrom E. coli JM109/pKSN2 is referred to as “B. coli pKSN2 extract”).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Reaction solutions of 30 μl were prepared and maintained for 1 hour at30° C. The reaction solutions consisted of a 0.1M potassium phosphatebuffer (pH7.0) containing 3 ppm of compound (II) labeled with ¹⁴C, 2 mMof β-NADPH (hereinafter, referred to as “component A”) (Oriental YeastCompany), 2 mg/ml of a ferredoxin derived from spinach (hereinafterreferred to as “component B”) (Sigma Company), 0.1 U/ml of ferredoxinreductase (hereinafter, referred to as “component C”) (Sigma Company)and 1841 of the supernatant fraction recovered in Example 12(2).Further, there were prepared and maintained similarly reaction solutionshaving no addition of at least one component utilized in the compositionof the above reaction solution, selected from component A, component Band component C. Three microliters (3 μl) of 2N HCl and 90 μl of ethylacetate were added and stirred into each of the reaction solutions afterthe maintenance. The resulting reaction solutions were centrifuged at8,000×g to recover 75 μl of the ethyl acetate layer. After drying theethyl acetate layers under reduced pressure, the residue was dissolvedin 6.0 μl of ethyl acetate. Five microliters (5.0 μl) thereof wasspotted to a silica gel TLC plate (TLC plate silica gel 60F₂₅₄, 20 cm×20cm, 0.25 mm thick, Merck Company). The TLC plate was developed with a6:1:2 mixture of chloroform, acetic acid and ethyl acetate for about 1hour. The solvents were then allowed to evaporate. The TLC plate wasexposed overnight to an imaging plate (Fuji Film Company). Next, theimaging plate was analyzed on Image Analyzer BAS2000 (Fuji FilmCompany). The presence of a spot corresponding to compound (III) labeledwith ¹⁴C were examined (Rf value 0.24 and 0.29). The results are shownin Table 11. TABLE 11 Reaction components component component componentE. coli compound (II) spot of A B C extract labeled with¹⁴C compound(III) + + + − + − + + + pKSN2 + − + + + pKSN671 + + − + + pKSN671 + − +− + pKSN671 + − + + − pKSN671 + + + + + pKSN671F + + − + + pKSN671F +− + − + pKSN671F + − + + − pKSN671F + +

Example 13 Obtaining the Present DNA (A9)

(1) Preparation of the Chromosomal DNA of Streptomyces carbophilusSANK62585

Streptomyces carbophilus SANK62585 (FERM BP-1145) was incubated withshaking at 30° C. for 1 day in 50 ml of YENS medium (0.3%(w/v) yeastextract, 0.5%(w/v) bacto-peptone, 0.3%(w/v) malt extract, 1.0%(w/v)glucose, 34%(w/v) sucrose and 0.2%(v/v) 2.5M MgCl₂ 6H₂O). The cells werethen recovered. The obtained cells were suspended in YEME mediumcontaining 1.4%(w/v) glycine and 60 mM EDTA and further incubated withshaking for a day. The cells were recovered from the culture medium.After washing once with distilled water, it was resuspended in buffer(100 mM Tris-HCl (pH8.0), 100 mM EDTA, 10 mM NaCl) at 1 ml per 200 mg ofthe cells. Two hundred micrograms per milliliter (200 μg/ml) ofegg-white lysozyme were added. The cell suspension was shaken at 30° C.for a hour. Further, 0.5% of SDS and 1 mg/ml of Proteinase K was added.The cell suspension was incubated at 55° C. for 3 hours. The cellsuspension was extracted twice with phenol.chloroform.isoamyl alcohol torecover each of the aqueous layers. Next, there was one extraction withchloroform.isoamyl alcohol to recover the aqueous layer. The chromosomalDNA was obtained by ethanol precipitating the aqueous layer.

(2) Isolation of the Present DNA (A9)

PCR was conducted by utilizing as the template the chromosomal DNAprepared in Example 13(1). As the primers, there was utilized thepairing of an oligonucleotide having the nucleotide sequence shown inSEQ ID NO: 74 and an oligonucleotide having the nucleotide sequenceshown in SEQ ID NO: 75 (hereinafter referred to as “primer paring 12”)or the pairing of an oligonucleotide having the nucleotide sequenceshown in SEQ ID NO: 76 and an oligonucleotide having the nucleotidesequence shown in SEQ ID NO; 77 (hereinafter referred to as “primerparing 13”). The PCR reaction solution amounted to 50 μl by adding the 2primers each amounting to 200 nM, 250 ng of the above chromosomal DNA, 4μl of dNTP mix (a mixture of 2.5 mM of each of the 4 types of dNTP), 5μl of lox ExTaq buffer, 0.5 μl of ExTaq polymerase (Takara ShuzoCompany) and distilled water. The reaction conditions of the PCR weremaintaining 95° C. for 2 minutes; repeating 30 cycles of a cycle thatincluded maintaining 97° C. for 15 seconds, followed by 60° C. for 30seconds, followed by 72° C. for 90 seconds, and then maintaining 72° C.for 4 minutes. After the maintenance, the reaction solution wassubjected to 0.8% agarose gel electrophoresis. The gel area containingthe DNA of about 500 bp was recovered from the gel subjected to the PCRreaction solution utilizing primer pairing 12. The gel area containingthe DNA of about 800 bp was recovered from the gel subjected to the PCRreaction solution utilizing primer pairing 13. The DNA were purifiedfrom each of the recovered gels by utilizing QIAquick gel extraction kit(Qiagen Company) according to the attached instructions. The obtainedDNA were ligated to the TA cloning vector pCR2.1 (Invitrogen Company)according to the instructions attached to said vector and was introducedinto E. Coli TOP10F′. The plasmid DNA were prepared from the obtained E.coli transformants, utilizing QIAprep Spin Miniprep Kit (QiagenCompany). A sequencing reaction was conducted with Dye terminator cyclesequencing FS ready reaction kit (Applied Biosystems Japan Company)according to the instructions attached to said kit, utilizing as primersthe oligonucleotide having the nucleotide sequence shown in SEQ ID NO:67and the oligonucleotide having the nucleotide sequence shown in SEQ IDNO: 68. The sequencing reaction utilized the obtained plasmid DNA as thetemplates. The reaction products were analyzed with a DNA sequencer 373A(Applied Biosystems Japan Company). As a result, the nucleotide sequenceshown in nucleotides 1 to 498 of the nucleotide sequence shown in SEQ IDNO: 78 was provided by the DNA obtained by the PCR utilizing primerpairing 12. The nucleotide sequence shown in nucleotides 469 to 1233 ofthe nucleotide sequence shown in SEQ ID NO: 78 was provided by the DNAobtained by the PCR utilizing primer pairing 13. The plasmid having thenucleotide sequence of nucleotides 1 to 498 shown in SEQ ID NO: 78 wasdesignated as pCRSCA1. The plasmid having the nucleotide sequence ofnucleotides 469 to 1233 shown in SEQ ID NO: 78 was designated aspCRSCA2.

Example 14 Expression of the Present Protein (A9) in E. Coli

(1) Production of a Transformed E. coli Having the Present DNA (A9)

With the plasmids obtained in Example 13(2), the above plasmid pCRSCA1was digested with NdeI and NcoI and pCRSCA2 was digested with NdeI andNcoI. The digestion products were subjected to agarose gelelectrophoresis. The gel area containing a DNA of about 500 bp was cutfrom the gel subjected to the digestion products of pCRSCA2. The gelarea containing a DNA of about 800 bp was cut from the gel subjected tothe digestion products of pCRSCA2. The DNA were purified from each ofthe recovered gels by utilizing QIAquick gel extraction kit (QiagenCompany) according to the attached instructions The 2 types of theobtained DNA were ligated together with the plasmid pKSN2 digested withNdeI and HindIII, utilizing ligation kit Ver.1 (Takara Shuzo Company) inaccordance with the instructions attached to said kit and introducedinto E. Coli JM109. The plasmid DNA was prepared from the obtained E.coli transformants. The structure thereof was analyzed. The plasmidcontaining the nucleotide sequence shown in SEQ ID NO; 78, in which theDNA encoding the present protein (A9) is inserted between the NdeI siteand the HindIII site of pKSN2 was designated as pKSNSCA.

(2) Expression of the Present Protein (A9) in E. coli and Recovery ofSaid Protein

E. coli JM109/pKSNSCA was cultured overnight at 37° C. in 10 ml of TBmedium (1.2%(w/v) tryptone, 2.4%(w/v) yeast extract, 0.4%(w/v) glycerol,17 mM potassium dihydrogenphosphate, 72 mM dipotassiumhydrogenphosphate) containing 50 μg/ml of ampicillin. The obtainedculture medium was transferred to 100 ml of TB medium containing 50μg/ml of ampicillin and cultured at 26° C., so that the OD660 was 0.2.When OD660 reached about 2.0, 5-aminolevulinic acid was added to thefinal concentration of 500 μM, and the culturing was continued. Thirty(30) minutes thereafter, IPTG was added to a final concentration of 200μM, and there was further culturing for 5 hours.

The cells were recovered from each of the culture mediums, washed with0.1M tris-HCl buffer (pH7.5) and suspended in 10 ml of said buffercontaining 1 mM PMSF. The obtained cell suspensions were subjected 6times to a sonicator (Sonifier (Branson Sonic Power Company)) at 3minutes each under the conditions of output 3, duty cycle 30%, in orderto obtain cell lysate solutions. After centrifuging the cell lysatesolutions (1,200×g, 5 minutes) the supernatants were recovered andcentrifuged (150,000×g, 70 minutes) to recover supernatant fractions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSNSCA is referred to as “E. coli pKSNSCA extract”).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Reaction solutions of 30 μl were prepared and maintained for 10 minutesat 30° C. The reaction solutions consisted of a 0.1M potassium phosphatebuffer (pH7.0) containing 3 ppm of compound (II) labeled with ¹⁴C, 2M ofβ-NADPH (hereinafter, referred to as “component A”) (Oriental YeastCompany), 2 mg/ml of a ferredoxin derived from spinach (hereinafterreferred to as “component B”) (Sigma Company), 0.1 U/ml of ferredoxinreductase (hereinafter, referred to as “component C”) (Sigma Company)and 18 μl of the supernatant fraction recovered in Example 14(2).Further, there were prepared and maintained similarly reaction solutionshaving no addition of at least one component utilized in the compositionof the above reaction solution, selected from component A, component Band component C. Three microliters (3 μl) of 2N HCl and 90 μl of ethylacetate were added and stirred into each of the reaction solutions afterthe maintenance. The resulting reaction solutions were centrifuged at8,000×g to recover 75 μl of the ethyl acetate layer. After drying theethyl acetate layers under reduced pressure, the residue was dissolvedin 6.0 μl of ethyl acetate. Five microliters (5.0 μl) thereof wasspotted to a silica gel TLC plate (TLC plate silica gel 60F₂₅₄, 20 cm×20cm, 0.25 mm thick, Merck Company). The TLC plate was developed with6:1:2 mixture of chloroform, acetic acid and ethyl acetate for about 1hour. The solvents were then allowed to evaporate. The TLC plate wasexposed overnight to an imaging plate (Fuji Film Company). Next, theimaging plate was analyzed on Image Analyzer BAS2000 (Fuji FilmCompany). The presence of a spot corresponding to compound (III) labeledwith ¹⁴C were examined (Rf value 0.24 and 0.29). The results are shownin Table 12. TABLE 12 Reaction components compound (II) componentcomponent component E. coli labeled with 14 spot of A B C extract Ccompound (III) + + + − + − + + + pKSNSCA + +

Example 15 Isolation of Soybean RuBPC Gene

After seeding soybean (cv. Jack), the soybean was cultivated at 27° C.for 30 days and the leaves were gathered. Two-tenths grams (0.2 g) to0.3 g of the gathered leaves were frozen with liquid nitrogen and weremilled with a mortar and pestle. Subsequently, the total RNA wasextracted from the milled product according to the manual attached withRNA extraction solvent ISOGEN (Nippon Gene Company). Further, cDNA wassynthesized with the use of Superscript First-strand Synthesis Systemfor RT-PCR (Invitrogen Company), by conducting the procedures inaccordance with the attached manual. Specifically, a 1st strand cDNA wassynthesized by utilizing the Oligo(dT)₁₂₋₁₈ primer provided by the kitas a primer and the total soybean RNA as the template and by addingthereto the reverse transcriptase provided by the kit. Next, there isamplified by PCR a DNA encoding the chloroplast transit peptide of thesmall subunit of ribulose-1,5-bisphosphate carboxylase (hereinafter, theribulose-1,5-bisphosphate carboxylase is referred to as “RuBPC”) ofsoybean (cv. Jack) followed by the 12 amino acids of a mature protein(hereinafter, the chloroplast transit peptide of the small subunit ofRuBPC of soybean (cv. Jack) is sometimes referred to as “rSt”; and theDNA encoding the chloroplast transit peptide of the small subunit ofRuBPC of soybean (cv. Jack) followed by the 12 amino acids of a matureprotein is referred to as “the present rSt12 DNA”). The PCR utilized theobtained cDNA as a template and as primers the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 86 and the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 87. The PCR utilizedLA Taq polymerase (Takara Shuzo Company). The PCR was conducted bymaintaining once 94° C. for 3 minutes; conducting 30 cycles of a cyclethat included maintaining 98° C. for 25 seconds and then 68° C. for 1minute; and maintaining once 72° C. for 10 minutes. Plasmid pCRrSt12(FIG. 5) was obtained by inserting the amplified DNA into thePCR-product cloning site of plasmid pCR2.1 (Invitrogen Company). Nextplasmid was introduced into the competent cells of E. coli JM109 strainand the ampicillin resistant strains were selected. Further, thenucleotide sequence of the plasmid contained in the selected ampicillinresistant strains was determined by utilizing the Dye Terminator CycleSequencing FS Ready Reaction kit (PE Applied Biosystems Company) and theDNA sequencer 373S (PE Applied Biosystems Company). As a result, thenucleotide sequence shown in SEQ ID NO: 88 was provided. It wasconfirmed that plasmid pCRrSt12 contained the present rSt12 DNA.

Example 16 Construction of a Chloroplast Expression Plasmid Containingthe Present Invention DNA (A1) for Direct Introduction

(1) Isolation of the Present Invention DNA (A1)

A DNA comprising the nucleotide sequence shown in SEQ ID NO: 6 wasamplified by PCR. The PCR was conducted by utilizing as the template thegenomic DNA of Actinomyces Streptomyces phaeochromogenes IFO12898 and byutilizing as primers the oligonucleotide consisting of the nucleotidesequence shown in SEQ ID NO: 93 and the oligonucleotide consisting ofthe nucleotide sequence shown in SEQ ID NO: 94 Further, a DNA comprisingthe nucleotide sequence shown in SEQ ID NO: 9 was amplified by PCR. ThePCR was conducted by utilizing as primers the oligonucleotide consistingof the nucleotide sequence shown in SEQ ID NO: 93 and theoligonucleotide sequence shown in SEQ ID NO: 95. Said PCR utilized theExpand High Fidelity PCR System (Boehringer Company). There wasconducted after maintaining once 97° C. for 2 minutes; conducting 10cycles of a cycle that included maintaining 97° C. for 15 seconds,followed by 60° C. for 30 seconds and followed by 72° C. for 1 minute;then conducting 15 cycles of a cycle that included maintaining 97° C.for 15 seconds, followed by 60° C. for 30 seconds and followed by 72° C.for 1 minute (wherein 20 seconds were added to the maintenance at 72° C.for each cycle); and then maintaining 72° C. for 7 minutes. PlasmidspCR657ET (FIG. 6) and pCR657FET (FIG. 7) were produced by inserting theamplified DNA into the PCR product cloning region of pCR2.1 (InvitrogenCompany). Furthermore, other than utilizing the oligonucleotideconsisting of the nucleotide sequence shown in SEQ ID NO: 96 and theoligonucleotide consisting of the nucleotide sequence shown in SEQ IDNO: 94, plasmid pCR657Bs (FIG. 8) was obtained with procedures similarto the method described above. Even further, other than utilizing theoligonucleotide consisting of the nucleotide sequence shown in SEQ IDNO: 96 and the oligonucleotide consisting of the nucleotide sequenceshown in SEQ ID NO 97, plasmid pCR657FBs (FIG. 9) was obtained withprocedures similar to the method described above. Next the plasmids wereintroduced into E. Coli DH5α competent cells (Takara Shuzo Company) andthe ampicillin resistant cells were selected. Further, the nucleotidesequences of the plasmids contained in the ampicillin resistant strainswere determined by utilizing BigDye Terminator Cycle Sequencing ReadyReaction kit v2.0 (PE Applied Biosystems Company) and DNA sequencer 3100(PE Applied Biosystems Company). As a result it was confirmed thatplasmids pCR657ET and pCR657Bs have the nucleotide sequence shown in SEQID NO: 6. It was confirmed that plasmids pCR657FET and pCR657FBs havethe nucleotide sequence shown in SEQ ID NO 9.

(2) Construction of a Chloroplast Expression Plasmid Having the PresentInvention DNA (A1) for Direct Introduction—Part (1)

A plasmid containing a chimeric DNA in which the present invention DNA(A1) was connected immediately after the nucleotide sequence encodingthe chloroplast transit peptide of soybean (cv. Jack) RuBPC smallsubunit (hereinafter sometimes referred to as the “sequence encoding thechloroplast transit peptide”) without a change of frames in the codonswas constructed as a plasmid for introducing the present invention DNA(A1) into a plant with the particle gun method.

First pCRrSt12 was digested with restriction enzyme HindIII and KpnI.The DNA comprising the present rSt12DNA was isolated. Further, a DNA ofabout 2640 bp was obtained by removing about a 40 bp DNA from plasmidvector pUC19 (Takara Shuzo Company) with a digestion with restrictionenzymes HindIII and KpnI. Next, the 5′ terminus of the DNA wasdephosphorylated with calf intestine alkaline phosphatase (Takara ShuzoCompany). The DNA containing the present rSt12DNA, obtained frompCRrSt12, was inserted thereto to obtain pUCrSt12 (FIG. 10). Next, DNAcomprising the present invention DNA (A1) were isolated by digestingeach of plasmids pCR657ET and pCR657FET with restriction enzymes EcoT22Iand SacI. Each of the obtained DNA was inserted between the EcoT22Irestriction site and the SacI restriction site of pUCrSt12 to obtainplasmids pUCrSt657 (FIG. 11) and pUCrSt657F (FIG. 12) containing achimeric DNA in which the present invention DNA (A1) was connectedimmediately after the nucleotide sequence encoding the chloroplasttransit peptide of soybean (cv. Jack) RuBPC small subunit without achange of frames in the codons.

pBICR16G6PT (described in Japanese unexamined patent 2000-166577) wasdigested with restriction enzyme EcoRI to isolate a DNA of about 3 kb.(Hereinafter, the promoter contained in the DNA described in the aboveJapanese unexamined patent is referred to as the “CR16G6 promoter”.Further, the terminator contained in the DNA described in the aboveJapanese unexamined patent is referred to as the “CR16 terminator”.)After digesting the plasmid vector pUC19 (Takara Shuzo Company) withrestriction enzyme EcoRI, the 5′ terminus of said DNA wasdephosphorylated with calf intestine alkaline phosphatase (Takara ShuzoCompany), The 3 kb DNA derived from pBICR16G6PT was inserted thereto toobtain plasmid pUCCR16G6-p/t (FIG. 13). pUCCR16G6-p/t was digested withrestriction enzymes HindIII and ScaI to isolate a DNA comprising theCR16G6 promoter. Further, by digesting plasmid vector pUC19 (TakaraShuzo Company) with restriction enzymes HindIII and EcoRI, a DNA of 51bp was removed and the remaining DNA consisting of 2635 bp was obtained.Next, the 5′ terminus of said DNA was dephosphorylated with calfintestine alkaline phosphatase (Takara Shuzo Company). The above DNAcomprising the CR16G6 promoter obtained from pUCCR16G6-p/t and aNotI-EcoRI linker (FIG. 14) obtained from annealing the oligonucleotideconsisting of the nucleotide sequence shown in SEQ ID No: 89 with theoligonucleotide consisting of the nucleotide sequence shown in SEQ IDNo: 90 were inserted thereto to obtain pUCCR12G6-p/tΔ (FIG. 15).pUCCR12G6-p/tΔ was digested with restriction enzymes NdeI and EcoRI toisolate a DNA having a partial nucleotide sequence of the CR16tterminator. Further, plasmid vector pUC19 (Takara Shuzo Company) wasdigested with restriction enzymes HindIII and EcoRI to obtain a DNA of2635 bp. The 5′ terminus of said DNA was dephosphorylated with calfintestine alkaline phosphatase (Takara Shuzo Company). The above DNAhaving a partial nucleotide sequence of the CR16t terminator obtainedfrom pUCCR12G6-p/tΔ and a HindIII-NotI linker (FIG. 16) obtained byannealing the oligonucleotide consisting of the nucleotide sequenceshown in SEQ ID NO: 91 with the oligonucleotide consisting of thenucleotide sequence shown in SEQ ID NO: 92 were inserted thereto toobtain pNdG6-ΔT (FIG. 17).

Next, by digesting each of plasmids pUCrSt657 and pUCr657F withrestriction enzymes BamHI and SacI, there was isolated the DNAcomprising a chimeric DNA in which the present invention DNA (A1) wasconnected immediately after the nucleotide sequence encoding thechloroplast transit peptide of soybean (cv. Jack) RuBPC small subunitwithout a change of frames in the codons. The DNA were inserted betweenthe restriction enzyme site of BglII and the restriction enzyme site ofSacI of plasmid pNdG6-ΔT to obtain each of plasmid pSUM-NdG6-rSt-657(FIG. 18) and plasmid pSUM-NdG6-rSt-657F (FIG. 19).

(3) Construction of a Chloroplast Expression Plasmid Having the PresentInvention DNA (A1) for Direct Introduction—Part (2)

A plasmid containing a chimeric DNA in which the present invention DNA(A1) was connected immediately after the nucleotide sequence encodingthe chloroplast transit peptide of soybean (cv. Jack) RuBPC smallsubunit and encoding thereafter 12 amino acids of the mature protein,without a change of frames in the codons was constructed as a plasmidfor introducing the present invention DNA (A1) into a plant with theparticle gun method. First, after digesting plasmid vector pKF19 (TakaraShuzo Company) with restriction enzyme BspHI, the DNA termini were bluntended by adding nucleotides to the double stranded gap, utilizing KODDNA polymerase (Toyobo Corporation). Plasmid pKF19ΔBs was obtained by aself-cyclizing the resulting DNA with T4 DNA ligase. The pCRrSt12obtained in Example 1 was digested with restriction enzyme HindIII andKpnI. The DNA comprising the present rSt12DNA was isolated. PlasmidpKF19ΔBs was digested with restriction enzymes HindIII and KpnI toobtain a DNA of about 2160 bp. The 5′ termini of said DNA weredephosphorylated with calf intestine alkaline phosphatase (Takara ShuzoCompany). The DNA comprising the present rSt12DNA obtained from pCRrSt12was inserted thereto to obtain pKFrSt12 (FIG. 20). Next, the plasmidspCR657Bs and pCR657FBs obtained in Example 16(1) were each digested withrestriction enzymes BspHI and SacI to isolate DNA comprising the presentinvention DNA (A1). Each of these DNA were inserted between therestriction site of BspHI and restriction site of SacI of plasmidpKFrSt12 to obtain plasmid pKFrSt12-657 (FIG. 21) and plasmidpKFrSt12457F (FIG. 22), which contained a chimeric DNA in which thepresent invention DNA (A1) was connected immediately after thenucleotide sequence encoding the chloroplast transit peptide of soybean(cv. Jack) RuBPC small subunit and encoding thereafter 12 amino acids ofthe mature protein, without a change of frames in the codons.

Next, each of plasmids pKFrSt12-657 and pKFrSt12-657F was digested withBamHI and SacI to obtain DNA comprising the present invention DNA (A1).Each of these DNA were inserted between the BglII restriction site andSacI restriction site of plasmid pNdG6-ΔT obtained in Example 16(2) toobtain plasmids pSUM-NdG6-rSt12-657 (FIG. 23) and pSUM-NdG6rSt12657F(FIG. 24) wherein the chimeric DNA, in which the present invention DNA(A1) was connected immediately after the nucleotide sequence encodingthe chloroplast transit peptide of soybean (cv. Jack) RuBPC smallsubunit and encoding thereafter 12 amino acids of the mature protein,without a change of frames in the codons, was connected downstream ofpromoter CR16G6.

Example 17 Introduction of the Present Invention DNA (A1) into Soybean

(1) Preparation of Proliferative Somatic Embryos

After dipping pods of soybeans (cultivar: Fayette and Jack) in 1% sodiumhypochlorite solution to sterilize, the immature seeds were taken out.The seed coat was exfoliated from the seed to remove the immature embryohaving a diameter of 2 to 5 mm. The embryonic axis of the obtainedimmature embryo was excised with a scalpel to prepare the immaturecotyledon. The immature cotyledon was divided into 2 cotyledon parts.Each cotyledon part was placed in the somatic embryo development medium,respectively. The somatic embryo development medium was a solidifiedmedium where 0.2%(w/v) Gelrite was added to Murashige-Skoog medium(described in Murashige T. and Skoog F., Physiol. Plant (1962) 15, p473;hereinafter referred to as “MS medium”) that was set to a pH of 7.0 andthat had 180 μM of 2,4-D and 30 g/L of sucrose added thereto. About 1month after the placement, the formed globular embryo was transplantedto the somatic embryo growth medium. The somatic embryo growth mediumwas a solidified medium where 0.2%(w/v) Gelrite was added to MS mediumthat was set to pH5.8 and that had 90 μM of 2,4-D and 30 μL of sucroseadded thereto. The globular embryo was thereafter transplanted to freshsomatic embryo growth medium 5 to 8 times at intervals of 2 to 3 weeks.Each of the culturing conditions utilizing the above somatic embryodevelopment medium and somatic embryo growth medium was 23 hours oflight with 1 hour of darkness and 23 to 25° C. for the whole day.

(2) Introduction of the Gene to Proliferative Somatic Embryos

After the globular embryo obtained in Example 17(1) is transplanted tofresh somatic embryo growth medium and cultured for 2 to 3 days, theglobular embryo was utilized to introduce the gene. PlasmidspSUM-NdG6-rSt657, pSUM-NdG6rSt657F, pSUM-NdG6-rSt12657 andpSUM-NdG6-rSt1265771 were coated onto Sold particles of a diameter of10.0 μm to conduct the gene introduction employing the particle gunmethod. The amount of the plasmids was 1.66 μg for 1 mg of the goldparticles. After introducing the gene, the embryo was cultured furtherfor 2 to 3 days. Each of the culturing conditions was 23 hours of lightwith 1 hour of darkness and 23 to 25° C. for the whole day.

(3) Selection of an Somatic Embryo with Hygromycin

The globular embryo after introducing the gene obtained in Example 17(2)was transplanted to an somatic embryo selection medium. The somaticembryo selection medium was a solidified medium where 0.2%(w/v) Gelriteand 15 mg/L of hygromycin were added to MS medium that was set to pH5.8and that had 90 μM of 2,4-D and 30 g/L of sucrose added thereto. Thesurviving globular embryo was thereafter transplanted to fresh somaticembryo selection medium 5 to 8 times at intervals of 2 to 3 weeks. Inthat time, the somatic embryo selection medium was a solidified mediumwhere 0.2%(w/v) Gelrite and 30 mg/L of hygromycin were added to MSmedium that was set to pH5.8 and that had 90 μM of 2,4-D and 30 g/L ofsucrose added thereto. Each of the culturing conditions utilizing theabove somatic embryo selection medium was 23 hours of light with 1 hourof darkness and 23 to 25° C. for the whole day.

(4) Selection of Somatic Embryo with Compound (II)

The globular embryo after introducing the gene obtained in Example 17(2)was transplanted to an somatic embryo selection medium. The somaticembryo selection medium was a solidified medium where 0.2%(w/v) Gelriteand 0.1 mg/L of compound (II) were added to MS medium that was set topH5.8 and that had 90 μM of 2,4-D and 30 g/L of sucrose added thereto.The surviving globular embryo was thereafter transplanted to freshsomatic embryo selection medium 5 to 8 times at intervals of 2 to 3weeks. In that time, the somatic embryo selection medium was asolidified medium where 0.2%(w/v) Gelrite and 0.3 to 1 mg/L of compound(II) were added to MS medium that was set to pH5.8 and that had 90 μM of2,4-D and 30 g/L of sucrose added thereto. Each of the culturingconditions utilizing the above somatic embryo selection medium was 23hours of light with 1 hour of darkness and 23 to 25° C. for the wholeday.

(5) Plant Regeneration from the Somatic Embryo

The globular embryos selected in Example 17(3) or 17(4) are transplantedto development medium and are cultured for 4 weeks in 23 hours of lightwith 1 hour of darkness and at 23 to 25° C. for the whole day. Thedevelopment medium is a solidified medium where 0.8% (w/v) of agar (WakoPure Chemical Industries, Ltd., use for plant tissue cultures) is addedto MS medium that is set to pH5.8 and that has 60 g/L of maltose addedthereto. White to yellow colored cotyledon-type embryos are obtained 6to 8 weeks thereafter. These cotyledon-type embryos are transplanted togermination medium and cultured for 2 weeks. The germination medium is asolidified medium where 0.2% (w/v) of Gelrite was added to MS mediumthat is set to pH5.8 and has 30 g/L of sucrose added thereto. As aresult, there can be obtained a soybean that has developed leaves andhas roots.

(6) Acclimation and Cultivation of the Regenerated Plant

The soybean obtained in Example 17(5) is transplanted to gardening soiland acclimated in an incubation chamber of 23 hours of light with 1 hourof darkness and 23 to 25° C. for the whole day. Two (2) weeksthereafter, the rooted plant is transferred to a pot having a diameterof 9 cm and cultivated at room temperature. The cultivation conditionsat room temperature are natural light conditions at 23° C. to 25° C. forthe whole day. Two to four (2 to 4) months thereafter, the soybean seedsare gathered.

(7) Evaluation of the Resistance to Herbicidal Compound (II)

Leaves of the regenerated plant are gathered and are split equally into2 pieces along the main vein. Compound (II) is spread onto the fullsurface of one of the leaf pieces. The other leaf piece is leftuntreated. These leaf pieces are placed on MS medium containing 0.8%agar and allowed to stand at room temperature for 7 days in light place.Then, each leaf piece is grounded with pestle and mortar in 5 ml of 80%aqueous acetone solution to extract chlorophyll. The extract liquid isdiluted 10 fold with 80% aqueous acetone solution and the absorbance ismeasured at 750 nm, 663 nm and 645 nm to calculate total chlorophyllcontent according to the method described by Mackenney G., J. Biol.Chem. (1941) 140, p 315. The degree of resistance to compound (II) canbe comparatively evaluated by showing in percentiles the totalchlorophyll content of the treated leaf piece with the total chlorophyllcontent of the untreated leaf piece.

Further, soil is packed into a plastic pot having a diameter of 10 cmand a depth of 10 cm. Seeds of the above described plant are seeded andcultivated in a greenhouse. An emulsion is prepared by mixing 5 parts ofcompound (II), 6 parts of sorpol3005X (Toho chemicals) and 89 parts ofxylene. A certain amount thereof was diluted with water containing 0.1%(v/v) of a sticking agent at a proportion of 1000 L for 1 hectare and isspread uniformly with a spray-gun onto the all sides of the foliage fromabove the plant cultivated in the above pot After cultivating the plantsfor 16 days in a greenhouse, the damage to the plants is investigated,and the resistance to compound (II) is evaluated.

Example 18 Construction of a Chloroplast Expression Plasmid Having thePresent Invention DNA (A1) for Agrobacterium Introduction

A plasmid for introducing the present invention DNA (A1) into a plantwith the agrobacterium method was constructed. First, after binaryplasmid vector pBI121 (Clontech Company) was digested with restrictionenzyme NotI, the DNA termini were blunt ended by adding nucleotides tothe double stranded gap, utilizing DNA polymerase I (Takara ShuzoCorporation). T4 DNA ligase was utilized for self-cyclization. After theobtained plasmid was digested with restriction enzyme EcoRI, the DNAtermini were blunt ended by adding nucleotides to the double strandedgap, utilizing DNA polymerase I (Takara Shuzo Corporation). T4 DNAligase was utilized for self-cyclization to obtain plasmidpBI121ΔNotIEcoRI. After digesting the plasmid with HindIII, the 5′ DNAterminus of the obtained DNA was dephosphorylated with calf intestinealkaline phosphatase (Takara Shuzo Company). A HindIII-NotI-EcoRI linker(FIG. 25) obtained by annealing the oligonucleotide consisting of thenucleotide sequence shown in SEQ ID NO: 98 with the oligonucleotideconsisting of the nucleotide sequence shown in SEQ ID NO: 99 wasinserted thereto. Binary plasmid vector pBI121 S (FIG. 26) was obtainedby self-cyclization. Said plasmid has a structure in which theHindIII-NotI-EcoRI linker was inserted in a direction in which theHindIII restriction site, the NotI restriction site, and the EcoRIrestriction site line up in turn from a location close to theβ-glucuronidase gene.

Next, each of plasmids pSUM-NdG6-rSt-657 and pSUM-NdG6-rSt-657F wasdigested with restriction enzymes HindIII and EcoRI, to obtain from eachthereof a chimeric DNA in which the present invention DNA (A1) wasconnected immediately after the nucleotide sequence encoding thechloroplast transit peptide of soybean (cv. Jack) RuBPC small subunitwithout a change of frames in the codons. These DNA were insertedbetween the HindIII restriction site and EcoRI restriction site of theabove binary plasmid vector pBI121S to obtain plasmids pBI-NdG6-rSt657(FIG. 27) and pBI-NdG6-rSt-657F (FIG. 28). Further, each of the aboveplasmids pSUM-NdG6-rSt12-657 and pSUM-NdG6rSt12-657F was digested withrestriction enzymes HindIII and EcoRI, to obtain from each a chimericDNA in which the present invention DNA (A1) was connected immediatelyafter the nucleotide sequence encoding the chloroplast transit peptideof soybean (cv. Jack) RuBPC small subunit and encoding thereafter 12amino acids of the mature protein, without a change of frames in thecodons. These DNA were inserted between the HindIII restriction site andEcoRI restriction site of the above binary plasmid vector pBI121S toobtain plasmids pBI-NdG6-rSt12-657 (FIG. 29) and pBI-NdG6-rSt12-657F(FIG. 30).

Example 19 Introduction of the Present Invention DNA (A1) to Tobacco

The present invention DNA (A1) was introduced into tobacco with theagrobacterium method, utilizing plasmid p]3]-NdG6-rSt-657, plasmidpBI-NdG6-rSt-657F, plasmid pBI-NdG6-rSt12657 and plasmidpBI-NdG6-rSt12-657F, obtained in Example 18.

First, the plasmids pBI-NdG6-rSt-657, pBI-NdG6-rSt657F,pBI-NdG6rSt12-657 and pBI-NdG6-rSt12-657F were introduced intoAgrobacterium tumefaciens LBA4404 (Clontech Company), respectively.Transformed agrobacterium strains bearing pBI-NdG6-rSt-657,pBI-NdG6-rSt-657F, pBI-NdG6-rSt12-657 or pBI-NdG6-rSt12-657F wereisolated by culturing the resultant transformants in LB agar medium(0.5% yeast extract, 1.0% Bacto tryptone, 0.5% NaCl) containing 300 mg/Lstreptomycin, 100 mg/L rifampicin and 25 mg/L kanamycin and by selectingthe resistant colonies.

Then, according to the method described in Manual for Gene Manipulationof Plant (by Hirofumi UCHIMIYA, Kodan-sha Scientific, 1992), the genewas introduced into tobacco. Agrobacterium strains bearing the aboveplasmids were each cultured at 28° C. overnight in LB medium containing300 mg/L streptomycin, 100 mg/L rifampicin and 25 mg/L kanamycin, andthen leaf pieces of tobacco (Nicotiana tabacum strain SR1) culturedsterilely were dipped in the liquid culture medium. The leaf pieces wereplanted and cultured at room temperature for 2 days in the light in MSagar medium (MS inorganic salts, MS vitamins, 3% sucrose and 0.8% agar;described in Murashige T. and Skoog F., Physiol. Plant. (1962) 15, p473) containing 0.1 mg/L of naphthalene acetic acid and 1.0 mg/L ofbenzyl aminopurine. Then, the leaf pieces were washed with sterilizedwater and cultured for 7 days on MS agar medium containing 0.1 mg/L ofnaphthalene acetic acid, 1.0 mg/L of benzyl aminopurine and 500 mg/L ofcefotaxime. Next, the leaf pieces were transplanted and cultured in MSagar medium containing 0.1 mg/L of naphthalene acetic acid, 1.0 mg/L ofbenzyl aminopurine, 500 mg/L of cefotaxime and 100 mg/L of kanamycin.The culture was conducted continuously for 4 months while transplantingthe leaf pieces to fresh medium of the same composition at intervals of4 weeks. At that time, the unfixed buds developing from the leave pieceswere transplanted and rooted in MS agar medium containing 300 mg/L ofcefotaxime and 50 mg/L of kanamycin to obtain regenerated bodies. Theregenerated bodies were transplanted to and cultured in MS agar mediumcontaining 50 mg/L of kanamycin to obtain, respectively, a transgenictobacco to which the T-DNA region of pBI-NdG6-rSt-657,pBI-NdG6-rSt-657F, pBI-NdG6-rSt12-657 or pBI-NdG6-rSt12-657F has beenintroduced.

Further, the plasmid pBI121S obtained in Example 18 was introduced intotobacco with the agrobacterium method. A transformed agrobacteriumstrain bearing pBI121S was isolated similarly to the above, other thanutilizing plasmid pBI1211S instead of pBI-NdG6-rSt-657,pBI-NdG6-rSt-657F, pBI-NdG6-rSt12-657 and pBI-NdG6-rSt12-6.57F. Next, atransgenic tobacco to which the T-DNA region of plasmid pBI121S has beenintroduced was obtained similarly to the above, utilizing saidtransformed agrobacterium.

Three (3) leaves were taken from the transgenic tobacco. Each leaf wasdivided into 4 pieces in which each piece was 5 to 7 mm wide. Each ofthe leaf pieces were planted onto MS agar medium containing 0.1 mg/L ofcompound (II) and cultured in the light at room temperature. On the 7thday of culturing, the herbicidal damage of each of the leaf pieces wasobserved. The leaf pieces derived from the tobacco to which the controlDNA (T-DNA region of plasmid pBI121 S) was introduced turned white andwithered. In contrast, the leaf pieces derived from the tobacco to whichthe present, invention DNA (A1) (the T-DNA region of plasmid ppBI-NdG6-rSt-657, plasmid pBI-NdG6-rSt12-657, pBI-NdG6-rSt-657F orpBI-NdG6-rSt12657F) was introduced grew continuously.

Example 20 Introduction of the Present Invention DNA into a Plant

Plasmids were constructed for introducing the present invention DNA (A2)with the particle gun method and the agrobacterium method. First, thepresent invention DNA (A2) having the nucleotide sequence shown in SEQID NO: 7 was amplified by PCR The PCR was conducted by utilizing as thetemplate the genomic DNA of Actinomyces Saccharopolyspora taberiJCM9383t and by utilizing as primers the oligonucleotide consisting ofthe nucleotide sequence shown in SEQ ID NO: 100 and the oligonucleotideconsisting of the nucleotide sequence shown in SEQ ID NO: 101. Said PCRutilized the Expand High Fidelity PCR System (Boehringer Company). Therewere conducted after maintaining once 97° C. for 2 minutes; repeating 10cycles of a cycle that included maintaining 97° C. for 15 seconds,followed by 60° C. for 30 seconds and followed by 72° C. for 60 seconds;then conducting 15 cycles of a cycle that included maintaining 97° C.for 15 seconds, followed by 60° C. for 30 seconds and followed by 72° C.for 1 minute (wherein 20 seconds were added to the maintenance at 72° C.for each cycle); and then maintaining 72° C. for 7 minutes. PlasmidspCR923Sp (FIG. 31) was produced by inserting the amplified DNA into thePCR product cloning region of pCR2.1-TOPO (Invitrogen Company). Next,the plasmid was introduced into E. Coli JM109 competent cells (TakaraShuzo Company) and the ampicillin resistant cells were selected.Further, the nucleotide sequences of the plasmids contained in theampicillin resistant stains were determined by utilizing BigDyeTerminator Cycle Sequencing Ready Reaction kit v2.0 (PE AppliedBiosystems Company) and DNA sequencer 373S (PE Applied BiosystemsCompany). As a result, it was confirmed that plasmid pCR923Sp has thenucleotide sequence shown in SEQ ID NO: 7.

Plasmid pKFrSt12, designed in Example 16(3), was digested withrestriction enzymes BamHI and SacI to isolate a DNA comprising thepresent rSt12DNA. Said DNA was inserted between the BglII restrictionsite and SphI restriction site of pNdG6-Δ T obtained in Example 16(2) toobtain plasmid pNdG6-rSt12 (FIG. 32). Plasmid pCR923Sp was digested withrestriction enzymes SphI and KpnI to obtain the DNA comprising thepresent invention DNA (A2). Plasmid pNdG6-rSt12 was digested withrestriction enzymes SphI and KpnI to remove the DNA encoding the 12amino acids of the mature protein of soybean (cv. Jack) RuBPC smallsubunit. In its place, the above DNA containing the present inventionDNA (A2) obtained from plasmid pCR923Sp was inserted to obtainpSUM-NdG6-rSt-923 (FIG. 33) wherein the CR16G6 promoter has connecteddownstream therefrom the chimeric DNA in which said DNA was connectedimmediately after the sequence encoding the chloroplast transit peptideof soybean (cv. Jack) RuBPC small subunit, without a change of frame inthe codons.

Next, plasmid pCR923Sp was digested with restriction enzyme SphI. Afterblunting the ends of the obtained DNA with KOD DNA polymerase, said DNAis further digested with restriction enzyme KpnI to isolate a DNAcontaining the present invention DNA (A2). Plasmid pKFrSt12 produced inExample 16(3) was digested with restriction enzyme BspHI. After bluntingthe ends of the obtained DNA with KOD DNA polymerase, said DNA isfurther digested with restriction enzyme KpnI to remove DNA of about 20bp. In its place, the above DNA containing the present invention DNA(A2) obtained from plasmid pCR923Sp was inserted to obtain plasmidpKFrSt12-923 (FIG. 34) comprising the chimeric DNA in which the presentinvention DNA (A2) was connected immediately after the nucleotidesequence encoding the chloroplast transit peptide of soybean (cv. Jack)RuBPC small subunit and encoding thereafter 12 amino acids of the matureprotein, without a change of frames in the codons. pKFrSt12-923 wasdigested with restriction enzymes SphI and KpnI to obtain the chimericDNA in which the present invention DNA (A2) and the DNA encoding thefirst 12 amino acids of the mature protein of soybean (cv. Jack) RuBPCsmall subunit are connected, Plasmid pNdG6-rSt12 was digested withrestriction enzymes SphI and KpnI to remove the DNA encoding the 12amino acids of the mature protein of soybean (cv. Jack) RuBPC smallsubunit. In its place, the above chimeric DNA obtained from plasmidpKFrSt12-923 was inserted to obtain plasmid pSUM-NdG6rSt12-923 (FIG. 35)in which the CR16G6 promoter has connected downstream therefrom thechimeric DNA in which said DNA containing the present invention DNA (A2)was connected immediately after the sequence encoding the chloroplasttransit peptide of soybean (cv. Jack) RuBPC small subunit and encodingthereafter 12 amino acids of the mature protein, without a change offrame in the codons.

The present invention DNA (A2) was introduced into soybean with theparticle gun method with the identical procedures of the methoddescribed in Example 17, utilizing the obtained plasmidspSUM-NdG6-rSt-923 and pSUM-NdG6-rSt12-923.

The above plasmid pSUM-NdG6-rSt-923 was digested with restrictionenzymes HindIII and EcoRI to isolate the DNA comprising the chimeric DNAin which said DNA containing the present invention DNA (A2) wasconnected immediately after the sequence encoding the chloroplasttransit peptide of soybean (cv. Jack) RuBPC small subunit, without achange of frame in the codons. As in producing pBI-NdG6-rSt657 inExample 18, the above DNA containing the chimeric DNA obtained fromplasmid pSUM-NdG6-rSt-923 was inserted between the HindIII restrictionsite and the EcoRI restriction site of binary vector pBI121S to obtainpBI-NdG6-rSt-923 (FIG. 36). Further, the above plasmidpSUM-NdG6-rSt12-923 was digested with HindIII and EcoRI, to isolate theDNA containing chimeric DNA in which said DNA containing the presentinvention DNA (A2) was connected immediately after the sequence encodingthe chloroplast transit peptide of soybean (cv. Jack) RuBPC smallsubunit and encoding thereafter 12 amino acids of the mature protein,without a change of frame in the codons. The chimeric DNA obtained frompSUM-NdG6-rSt12-923 was inserted between the HindIII restriction siteand EcoRI restriction sites of binary vector pBI121S to obtainpBI-NdG6-rSt12-923 (FIG. 37).

Each of the plasmids pBI-NdG6-rSt-923 and pBI-NdG6rSt12-923 wasintroduced into Agrobacterium tumefaciens LBA4404. The resultanttransformants were cultured in LB medium containing 300 μg/ml ofstreptomycin, 100 μg/ml of rifampicin and 25 μg/ml of kanamycin. Thetransformants were selected to isolate agrobacterium strains bearingpBI-NdG6-rSt-923 or pBI-NdG6-rSt12-923.

Leaf pieces of sterily cultured tobacco were infected with each of theagrobacterium strain bearing pBI-NdG6-rSt-923 and the agrobacteriumstrain bearing pBI-NdG6-rSt12-923. Tobaccos in which the presentinvention DNA (A2) has been introduced were obtained under theprocedures similar to the methods described in Example 19.

Three (3) leaves were taken from the obtained transgenic tobacco. Eachleaf was divided into 4 pieces in which each piece was 5 to 7 mm wide.Each of the leaf pieces were planted onto MS agar medium containing 0.1mg/L of compound (II) and cultured in the light at room temperature. Onthe 7th day of culturing, the herbicidal damage of each of the leafpieces was observed. The leaf pieces derived from the tobacco to whichthe control DNA (T-DNA region of plasmid pBI121S) was introduced turnedwhite and withered. In contrast, the leaf pieces derived from thetobacco to which the present invention DNA (A2) (the T-DNA region ofplasmid pBI-NdG6-rSt923 or plasmid pBI-NdG6-rSt12-923) was introducedgrew continuously.

Example 21 Introduction of the Present Invention DNA (A3) into Tobacco

Plasmids were constructed for introducing the present invention DNA (A3)into a plant with the particle gun method and with the agrobacteriummethod.

First, the present invention DNA (A3) having the nucleotide sequenceshown in SEQ ID NO: 8 was amplified by PCR. The PCR was conducted byutilizing as the template the genomic DNA of Actinomyces Streptomycestestaceus ATCC21469 and by utilizing as primers the oligonucleotideconsisting of the nucleotide sequence shown in SEQ ID NO: 102 and theoligonucleotide consisting of the nucleotide sequence shown in SEQ IDNO: 103. Said PCR utilized the Expand High Fidelity PCR System(Boehringer Company). There were conducted after maintaining once 97° C.for 2 minutes; repeating 10 cycles of a cycle that included maintaining97° C. for 15 seconds, followed by 60° C. for 30 seconds and followed by72° C. for 1 minute; then conducting 15 cycles of a cycle that includedmaintaining 97° C. for 15 seconds, followed by 60° C. for 30 seconds andfollowed by 72° C. for 1 minute (wherein 20 seconds were added to themaintenance at 72° C. for each cycle); and then maintaining once 72° C.for 7 minutes. Plasmid pCR671ET (FIG. 38) was produced by inserting theamplified DNA into the PCR product cloning region of pCR2.1 (InvitrogenCompany). Further, plasmid pCR671Bs (FIG. 39) was obtained with theprocedures similar to the method described above, other than utilizingas the PCR primers, the oligonucleotide consisting of the nucleotidesequence shown in SEQ ID NO: 104 and the oligonucleotide consisting ofthe nucleotide sequence shown in SEQ ID NO: 103. Next, the plasmids wereintroduced into E. Coli JM109 competent cells (Takara Shuzo Company) andthe ampicillin resistant cells were selected. Further, the nucleotidesequences of the plasmids contained in the ampicillin resistant strainswere determined by utilizing BigDye Terminator Cycle Sequencing ReadyReaction kit v2.0 (PE Applied Biosystems Company) and DNA sequencer 3100(PE Applied Biosystems Company). As a result, it was confirmed thatplasmids pCR671ET and pCR671Bs have the nucleotide sequence shown in SEQID NO: 8.

Plasmid pCR671ET was digested with restriction enzymes EcoT22I and KpnIto isolate DNA comprising the present invention DNA (A3). Said DNA wasinserted between the EcoT22 I restriction site and the KpnI restrictionsite to obtain plasmid pUCrSt671 (FIG. 40) comprising the chimeric DNAin which the present invention DNA (A3) was connected immediately afterthe sequence encoding the chloroplast transit peptide of soybean (cv.Jack) RuBPC small subunit, without a change of frame in the codons.Plasmid pUCrSt671 was digested with restriction enzymes NheI and KpnI toisolate DNA comprising the present invention DNA (A3). PlasmidpNdG6-rSt12, obtained in Example 16(2), was digested with restrictionenzymes NheI and KpnI to remove DNA of about 80 bp. In its place, theabove DNA containing the present invention DNA (A3) obtained fromplasmid pUCrSt671 was inserted to obtain pSUM-NdG6-rSt-671 (FIG. 41)wherein the CR16G6 promoter has connected downstream therefrom thechimeric DNA in which the present invention DNA (A3) was connectedimmediately after the sequence encoding the chloroplast transit peptideof soybean (cv. Jack) RuBPC small subunit, without a change of frame inthe codons.

Plasmid pCR671Bs was digested with restriction enzymes BspHI and KpnI toisolate a DNA comprising the present invention DNA (A3). Said DNA wasinserted between the BspHI restriction site and KpnI restriction site ofpKFrSt12 obtained in Example 16(3) to obtain plasmid pKFrSt12-671 (FIG.42) containing the chimeric DNA in which the present invention DNA (A3)was connected immediately after the sequence encoding the chloroplasttransit peptide of soybean (cv. Jack) RuBPC small subunit and encodingthereafter 12 amino acids of the mature protein, without a change offrame in the codons. Plasmid pNdG6-rSt12 obtained in Example 20 wasdigested with restriction enzymes NheI and KpnI to remove DNA of about80 bp. In its place, the above DNA containing the present invention DNA(A3) obtained from plasmid pKFrSt12-671 was inserted to obtainpSUM-NdG6-rSt12-671 (FIG. 43) wherein the CR16G6 promoter has connecteddownstream therefrom the chimeric DNA in which the present invention DNA(A3) was connected immediately after the sequence encoding thechloroplast transit peptide of soybean (cv. Jack) RuBPC small subunitand encoding thereafter 12 amino acids of the mature protein, without achange of frame in the codons.

The present invention DNA (A3) was introduced into soybean with theparticle gun method with procedures similar to the method described inExample 17, utilizing the obtained plasmids pSUM-NdG6-rSt471 andpSUM-NdG6-rSt12-671.

The above plasmid pSUM-NdG6-rSt671 was digested with restriction enzymesHindIII and EcoRI to isolate the chimeric DNA in which the presentinvention DNA (A3) was connected immediately after the sequence encodingthe chloroplast transit peptide of soybean (cv. lack) RuBPC smallsubunit without a change of frame in the codons. The above DNAcontaining the chimeric DNA obtained from plasmid pSUM-NdG6-rSt-671 wasinserted between the HindIII restriction site and the EcoRI restrictionsite of binary vector plasmid pBI121S obtained in Example 18, to obtainp]31-NdG6-rSt-671 (FIG. 44). Further, the above plasmidpSUM-NdG6-rSt12-671 was digested with restriction enzymes HindIII andEcoRI, to isolate the DNA containing chimeric DNA in which said DNAcontaining the present invention DNA (A3) was connected immediatelyafter the sequence encoding the chloroplast transit peptide of soybean(cv. Jack) RuBPC small subunit and encoding thereafter 12 amino acids ofthe mature protein, without a change of flame in the codons. Thechimeric DNA obtained from pSUM-NdG6-rSt12-671 was inserted between theHindIII restriction site and EcoRI restriction sites of binary plasmidvector pBI121S to obtain pBI-NdG6-rSt12671 (FIG. 45).

Each of the plasmids pBI-NdG6-rSt-671 and pBI-NdG6-rSt12671 wereintroduced into Agrobacterium tumefaciens LBA4404. The resultanttransformants were cultured in LB medium containing 300 μg/ml ofstreptomycin, 100 μg/ml of rifampicin and 25 μg/ml of kanamycin. Thetransformants were selected to isolate agrobacterium strains bearingpBI-NdG6-rSt-671 or pBI-NdG6-rSt12671.

Leaf pieces of sterily cultured tobacco were infected with each of theagrobacterium strain bearing pBI-NdG6-rSt-671 and the agrobacteriumstrain bearing pBI-NdG6-rSt12-671. Tobaccos in which the presentinvention DNA (A3) has been introduced were obtained under theprocedures similar to the methods described in Example 19.

Three (3) leaves are taken from the transgenic tobaccos. Each leaf isdivided into 4 pieces in which each piece was 5 to 7 mm wide. Each ofthe leaf pieces are planted onto MS agar medium containing 0.1 mg/L ofcompound (II) and cultured in the light at room temperature. On the 7thday of culturing, the herbicidal damage of each of the leaf pieces isobserved.

Example 22 Expression of the Present Invention Protein (B1) in E. Coli

(1) Production of a Transformed E. coli of the Present Invention DNA(B1)

PCR was conducted by utilizing as a template the chromosomal DNAprepared from Streptomyces phaeochromogenes IFO12898 in Example 3(1).The PCR reaction solution amounted to 50 μl by adding 300 ng of theabove chromosomal DNA, 4 μl of dNTP mix (a mixture of 25 mM of each ofthe 4 types of dNTP), 5 μl of 10× ExTaq buffer, 0.5 μl of ExTaqpolymerase (Takara Shuzo Company), distilled water and 200 nM of each ofthe oligonucleotide having the nucleotide sequence shown in SEQ ID NO:105 and the oligonucleotide having the nucleotide sequence shown in SEQID NO: 53. The reaction conditions of the PCR were after maintaining 97°C. for 2 minutes; repeating 25 cycles of a cycle that includedmaintaining 97° C. for 15 seconds, followed by 60° C. for 30 seconds andfollowed by 72° C. for 90 seconds; and then maintaining 72° C. for 4minutes. The reaction solution after the maintenance and the vectorpCR2.1-TOPO (Invitrogen Company) were ligated according to theinstructions attached to said vector and were introduced into E. ColiTOP10F′. The plasmid DNA were prepared from the obtained E. colitransformants, utilizing QIAprep Spin Miniprep Kit (Qiagen Company).Sequencing reactions were conducted with Dye terminator cycle sequencingFS ready reaction kit (Applied Biosystems Japan Company) according tothe instructions attached to said kit utilizing as primers theoligonucleotide consisting of the nucleotide sequence shown in SEQ IDNO: 67 and the oligonucleotide consisting of the nucleotide sequenceshown in SEQ ID NO: 68. The sequencing reactions utilized the obtainedplasmid DNA as the template. The reaction products were analyzed with aDNA sequencer 373A (Applied Biosystems Japan Company) Based on theresults, the plasmid having the nucleotide sequence shown in SEQ ID NO:15 was designated as pCR657FD.

Next, pCR657FD was digested with restriction enzymes NdeI and HindIII.The digestion products were subjected to agarose gel electrophoresis.The gel area containing a DNA of about 200 bp was cut from the gel. TheDNA was purified from the recovered gels by utilizing QIA quick gelextraction kit (Qiagen Company) according to the attached instructions.The obtained DNA and the plasmid pKSN2 digested with NdeI and HindIIIwere ligated with ligation kit Ver.1 (Takara Shuzo Company) according tothe instructions attached to said kit and introduced into E. Coli JM109.The plasmid DNA were prepared from the obtained E. coli transformants.The structures thereof were analyzed. The plasmid containing thenucleotide sequence shown in SEQ ID NO: 15, in which the DNA of about200 bp encoding the present invention protein (B1) is inserted betweenthe NdeI site and the HindIII site of pKSN2 was designated as pKSN657FD.The plasmid pKSN657FD was introduced into E. coli JM109. The obtained E.coli transformant was designated JM109/pKSN657FD. Further, plasmid pKSN2was introduced into E. coli JM109. The obtained E. coli transformant wasdesignated as JM109/pKSN2.

(2) Expression of the Present Invention Protein (B1) in E. coli andRecovery of said Protein

E. coli JM109/pKSN657FD and E. Coli JM109/pKSN2 were each culturedovernight at 37° C. in 10 ml of TB medium (1.2%(w/v) tryptone, 2.4%(w/v)yeast extract, 0.4%(w/v) glycerol, 17 mM potassium dihydrogenphosphate,72 mM dipotassium hydrogenphosphate) containing 50 μg/ml of ampicillin.A milliliter (1 ml) of the obtained culture medium was transferred to100 ml of TB medium containing 50 μg/ml of ampicillin and cultured at26° C. Thirty (30) minutes after the OD660 reached about 05, IPTG wasadded to a final concentration of 1 mM, and there was further culturingfor 20 hours.

The cells were recovered from each of the culture mediums, washed with0.1M tris-HCl buffer (pH7.5) and suspended in 10 ml of said buffercontaining 1 mM PMSF. The obtained cell suspensions were subjected 6times to a sonicator (Sonifier (Branson Sonic Power Company)) at 3minutes each under the conditions of output 3, duty cycle 30%, in orderto obtain cell lysate solutions. After centrifuging the cell lysatesolutions (1,200×g, 5 minutes) the supernatants were recovered andcentrifuged (150,000×g, 70 minutes) to recover supernatant fractions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN657FD is referred to as “E. coli pKSN657FD extract” and thesupernatant fraction obtained from E. coli JM109/pKSN2 is referred to as“E. coli pKSN2 extract”). A microliter (1 μl) of the above supernatantfractions was analyzed on a 15% to 25% SDS-PAGE and stained with CBB. Asa result, notably more intense bands were identified in the E. colipKSN657FD extract than the E. coli pKSN2 extract, at the electrophoresislocations corresponding to the molecular weight of 7 kDa. It was shownthat E. coli JM109/pKSN657FD expressed the present invention protein(B1).

(3) Use of the Present Invention Protein (B1) for a Reaction System ofConverting Compound (II) to Compound (III)

Reaction solutions of 30 μl were prepared and maintained for 10 minutesat 30° C. The reaction solutions consisted of a 0.1M potassium phosphatebuffer (p17.0) containing 3 ppm of compound (II) labeled with ¹⁴C, 2 mMof β-NADPH (hereinafter, referred to as “component A”) (Oriental YeastCompany), 9 μl of the E. coli pKSN657FD extract recovered in Example22(2), 0.1 U/ml of ferredoxin reductase (hereinafter, referred to as“component C”) (Sigma Company) and 15 μl of the E. coli pKSN657F extractrecovered in Example 4(2) (hereinafter referred to as “component D”).Further, there were prepared reaction solutions in which 2 mg/ml offerredoxin derived from spinach (hereinafter referred to as “componentB”) (Sigma Company) was added in the place of the E. coli pKSN657FDextract and a reaction solution in which nothing was added in the placeof the E. coli pKSN657FD extract. Such reaction solutions weremaintained similarly. Three microliters (3 μl) of 2N HCl and 90 μl ofethyl acetate were added and mixed into each of the reaction solutionsafter the maintenance. The resulting reaction solutions were centrifugedat 8,000×g to recover 75 μl of the ethyl acetate layer. After drying theethyl acetate layers under reduced pressure, the residue was dissolvedin 6.0 μl of ethyl acetate. Five microliters (5.0 μl) thereof wasspotted to a silica gel TLC plate (TLC plate silica gel 60F₂₅₄, 20 cm×20cm, 0.25 mm thick, Merck Company). The TLC plate was developed with a6:1:2 mixture of chloroform, acetic acid and ethyl acetate for about 1hour. The solvents were then allowed to evaporate. The TLC plate wasexposed overnight to an imaging plate (Fuji Film Company). Next, theimaging plate was analyzed on Image Analyzer BAS2000 (Fuji FilmCompany). The presence of a spot corresponding to compound (A1) labeledwith ¹⁴C were examined (Rf value 0.24 and 0.29). The results are shownin Table 13. TABLE 13 Reaction components spot of component E. colicomponent component component compound (II) compound A extract B C Dlabeled with¹⁴C (III) + pKSN657FD − + + + + + − − + + + − + − + + + + +

Example 23 Expression of the Present Invention Protein (B2) in E. Coli

(1) Production of a Transformed E. coli Having the Present Invention DNA(B2)

PCR is conducted by utilizing as a template the chromosomal DNA preparedfrom Saccharopolyspora taberi JCM9383t in Example 6(1). The PCR reactionsolution amounts to 50 μl by adding 300 ng of the above chromosomal DNA,4 μl of dNTP mix (a mixture of 2.5 mM of each of the 4 types of dNTP), 5μl of 10× ExTaq buffer, 0.5 μl of ExTaq polymerase (Takara ShuzoCompany), distilled water and 200 nM of each of the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 106 and theoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 63.The reaction conditions of the PCR are after maintaining 97° C. for 2minutes; repeating 25 cycles of a cycle that included maintaining 97° C.for 15 seconds, followed by 60° C. for 30 seconds and followed by 72° C.for 90 seconds; and then maintaining 72° C. for 4 minutes. The reactionsolution after the maintenance and the vector pCR2.1-TOPO (InvitrogenCompany) are ligated according to the instructions attached to saidvector and introduced into E. Coli TOP10F′. The plasmid DNA are preparedfrom the obtained E. coli transformants, utilizing QIAprep Spin MiniprepKit (Qiagen Company), Sequencing reactions are conducted with Dyeterminator cycle sequencing FS ready reaction kit (Applied BiosystemsJapan Company) according to the instructions attached to said kit,utilizing as primers the oligonucleotide consisting of the nucleotidesequence shown in SEQ ID NO: 67 and the oligonucleotide consisting ofthe nucleotide sequence shown in SEQ ID NO: 68. The sequencing reactionsutilize the obtained plasmid DNA as the template. The reaction productsare analyzed with a DNA sequencer 373A (Applied Biosystems JapanCompany). Based on the results, the plasmid having the nucleotidesequence shown in SEQ ID NO: 16 is designated as pCR923FD.

Next plasmid pCR923FD is digested with restriction enzymes NdeI andHindIII. The digestion products are subjected to agarose gelelectrophoresis. The gel area containing a DNA of about 200 bp is cutfrom the gel. The DNA is purified from the recovered gels by utilizingQIA quick gel extraction kit (Qiagen Company) according to the attachedinstructions. The obtained DNA and the plasmid pKSN2 digested with NdeIand HindIII are ligated with ligation kit Ver.1 (Takara Shuzo Company)according to the instructions attached to said kit and introduced intoE. Coli JM109. The plasmid DNA are prepared from the obtained E. colitransformants. The structures thereof are analyzed. The plasmidcontaining the nucleotide sequence shown in SEQ ID NO: 16, in which theDNA of about 200 bp encoding the present invention protein (B2) isinserted between the NdeI site and the HindIII site of pKSN2 isdesignated as pKSN923FD. The plasmid pKSN923FD is introduced into E.coli JM109. The obtained E. coli transformant is designated asJM109/pKSN923FD. Further, plasmid pKSN2 is introduced into E. coliJM109. The obtained E. coli transformant is designated as JM109/pKSN2.

(2) Expression of the Present Invention Protein (B2) in E. coli andRecovery of Said Protein

E. coli JM109/pKSN923FD and E. Coli JM109/pKSN2 are each culturedovernight at 37° C. in 10 ml of TB medium (1.2%(w/v) tryptone, 2.4%(w/v)yeast extract, 0.4%(w/v) glycerol, 17 mM potassium dihydrogenphosphate,72 mM of dipotassium hydrogenphosphate) containing 50 μg/ml ofampicillin. A milliliter (1 ml) of the obtained culture medium istransferred to 100 ml of TB medium containing 50 μg/ml of ampicillin andcultured at 26° C. Thirty (30) minutes after the OD660 reached about0.5, IPTG is added to a final concentration of 1 mM, and there isfurther culturing for 20 hours.

The cells are recovered from each of the culture mediums, washed with0.1M tris-HCl buffer (pH7.5) and suspended in 10 ml of said buffercontaining 1 mM PMSF. The obtained cell suspensions are subjected 6times to a sonicator (Sonifier (Branson Sonic Power Company)) at 3minutes each under the conditions of output 3, duty cycle 30%, in orderto obtain cell lysate solutions. After centrifuging the cell lysatesolutions (1,200×g, 5 minutes) the supernatants are recovered andcentrifuged (150,000×g, 70 minutes) to recover supernatant fractions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN923FD is referred to as “E. coli pKSN923FD extract” and thesupernatant fraction obtained from E. coli JM109/pKSN2 is referred to as“E. coli pKSN2 extract”). A microliter (1 μl) of the above supernatantfractions is analyzed on a 15% to 25% SDS-PAGE and stained with CBB. Bydetecting notably more intense bands in the E. coli pKSN923FD extractthan the E. coli pKSN2 extract, at the electrophoresis locationscorresponding to the molecular weight of 7 kDa, it is possible toconfirm to E. coli expression of the present invention protein (B2).

(3) Use of the Present Invention Protein (B2) for a Reaction System ofConverting Compound (II) to Compound (III)

Reaction solutions of 30 μl are prepared and maintained for 10 minutesat 30° C. The reaction solutions consist of a 0.1M potassium phosphatebuffer (pH7.0) containing 3 ppm of compound (II) labeled with ¹⁴C, 2 mMof β-NADPH (hereinafter, referred to as “component A”) (Oriental YeastCompany), 9 μl of the E. coli pKSN923FD extract recovered in Example23(3), 0.1 U/ml of ferredoxin reductase (hereinafter, referred to as“component C”) (Sigma Company) and 15 μl of the E. coli pKSN657F extractrecovered in Example 4(2) (hereinafter referred to as “component D)”).Further, there are prepared reaction solutions in which 2 mg/ml offerredoxin derived from spinach (hereinafter referred to as “componentB”) (Sigma Company) is added in the place of the E. coli pKSN923FDextract and a reaction solution in which nothing is added in the placeof the E. coli pKSN923FD extract. Such reaction solutions are maintainedsimilarly. Three microliters (3 μl) of 2N HCl and 90 μl of ethyl acetateare added and mixed into each of the reaction solutions after themaintenance. The resulting reaction solutions are centrifuged at 8,000×gto recover 75 μl of the ethyl acetate layer. After drying the ethylacetate layers under reduced pressure, the residue is dissolved in 6.0μl of ethyl acetate. Five microliters (5.0 μl) thereof is spotted to asilica gel TLC plate (TLC plate silica gel 60F₂₅₄, 20 cm×20 cm, 0.25 mmthick, Merck Company). The TLC plate is developed with a 6:1:2 mixtureof chloroform, acetic acid and ethyl acetate for about 1 hour. Thesolvents are then allowed to evaporate. The TLC plate is exposedovernight to an imaging plate (Fuji Film Company). Next, the imagingplate is analyzed on Image Analyzer BAS2000 (Fuji Film Company). Thepresence of a spot corresponding to compound (III) labeled with ¹⁴C areexamined (Rf value 0.24 and 0.29). By confirming that compound (III) isproduced in the reaction including component A, E. coli pKSN923FDextract, component C and component D, it can be confirmed that thepresent invention protein (B2) can be used instead of the ferredoxinderived from spinach in a reaction system of converting compound (II) tocompound (III).

Example 24 Expression of the Present Invention Protein (B3) in E. Coli

(1) Production of a Transformed E. coli Having the Present Invention DNA(B3)

PCR is conducted similarly to the methods described in Example 23(1),other than utilizing as a template the chromosomal DNA prepared fromStreptomyces testaceus ATCC 21469 in Example 11(1) and utilizing as theprimers the oligonucleotide having the nucleotide sequence shown in SEQID NO: 107 and the oligonucleotide having the nucleotide sequence shownin SEQ ID NO: 72. Plasmid pCR671FD having the nucleotide sequence shownin SEQ ID NO: 17 is obtained similarly to the method described inExample 23(1) utilizing the obtained reaction solution.

Next, utilizing said plasmid, plasmid pKSN671fD in which the presentinvention DNA (B3) is inserted between the NdeI site and HindIII site ofpKSN2 is obtained similarly to the method described in Example 23(1). Byintroducing the plasmid into E. coli JM109, E. coli JM109/pKSN671FDhaving the present invention DNA (B3) can be obtained.

(2) Expression of the Present Invention Protein (B3) in E. coli andRecovery of Said Protein

Utilizing E. coli JM109/pKSN671FD, supernatant fractions (hereinafterreferred to as “E. coli pKSN671 FD extract”) are recovered similarly tothe method described in Example 23(2). A microliter (1 μl) of the abovesupernatant fractions is analyzed on a 15% to 25% SDS-PAGE and stainedwith CBB. As a result, by detecting notably more intense bands in the E.coli pKSN671FD extract than the E. coli pKSN2 extract, at theelectrophoresis location corresponding to the molecular weight of 7 kDa,it is possible to confirm the expression of the present inventionprotein (B3) in E. coli.

(3) Use of the Present Invention Protein (B3) for a Reaction System ofConverting Compound (II) to Compound (III)

Other than utilizing E. coli pKSN671FD extract recovered in Example24(2), the spot corresponding to compound (III) labeled with ¹⁴C (Rfvalues 0.24 and 0.29) is confirmed similarly to the method described inExample 23(3) By confirming that compound (III) is produced in thereaction including component A, E. coli pKSN671FD extract, component Cand component D, it can be confirmed that the present invention protein(B3) can be used instead of the ferredoxin derived from spinach in areaction system of converting compound (II) to compound (III).

Example 25 Preparation of the Present Invention Protein (A4)

(1) Preparation of the Crude Cell Extract

A frozen stock of Streptomyces achromogenes IFO12735 was added to 10 mlof A medium (0.1%(w/v) of glucose, 0.5%(w/v) tryptone, 0.5%(w/v) yeastextract, 0.1%(w/v) of dipotassium hydrogenphosphate, pH7.0) in a largetest tube and incubated with shaking at 30° C. for 1 day to obtain apre-culture. Eight milliliters (8 ml) of the pre-culture was added to200 ml of A medium and was incubated with rotary shaking in a 500 mlbaffled flask at 30° C. for 2 days. Cell pellets were recovered bycentrifuging (3,000×g, 10 min.) the resulting culture. These cellpellets were suspended in 100 ml of B medium (1%(w/v) glucose, 0.1% beefextract, 0.2%(w/v) tryptose) containing compound (II) at 100 ppm andwere incubated with reciprocal shaking in a 500 ml Sakaguchi flask for20 hours at 30° C. Cell pellets were recovered by centrifuging (3,000×g,10 min.) 2 L of the resulting culture. The resulting cell pellets werewashed twice with 1 L of 0.1M potassium phosphate buffer (pH7.0) toprovide 136 g of the cell pellets.

These cell pellets were suspended in 0.1M potassium phosphate buffer(pH7.0) at 1 ml to 2 ml for 1 g of the cell pellets. A millimolar of (1mM) PMSF, 5 mM of benzamidine HCl, 1 mM of EDTA, 3 μg/ml of leupeptin, 3μg/ml of pepstatin and 1 mM of dithiotritol were added to the cellsuspension. A cell lysate solution was obtained by disrupting twicerepetitively the suspension with a French press (1000 kg/cm²) (OhtakeSeisakusho). After centrifuging the cell lysate solution (40,000×g, 30minutes), the supernatant was recovered and centrifuged for 1 hour at150,000×g to recover the supernatant (hereinafter referred to as the“crude cell extract”).

(2) Determination of the Ability of Converting Compound (II) to Compound(III)

There was prepared 30 μl of a reaction solution consisting of 0.1Mpotassium phosphate buffer (pH7.0) containing 3 ppm of compound (II)labeled with ¹⁴C, 2.4 mM of β-NADPH (hereinafter, referred to as“component A”) (Oriental Yeast Company), 0.5 mg/ml of a ferredoxinderived from spinach (hereinafter referred to as “component B”) (SigmaCompany), 1 U/ml of ferredoxin reductase (hereinafter, referred to as“component C”) (Sigma Company) and 15 μl of the crude cell extractrecovered in Example 25(1). The reaction solution was maintained at 30°C. for a hour. Further, there was prepared and maintained similarly areaction solution having no addition of at least one component utilizedin the composition of the above reaction solution, selected fromcomponent A, component B and component C. Three microliters (3 μl) of 2NHCl and 90 μl of ethyl acetate were added and mixed into each of thereaction solutions after the maintenance. The resulting reactionsolutions were centrifuged at 8,000×g to recover 75 μl of the ethylacetate layer. After drying the ethyl acetate layers under reducedpressure, the residue was dissolved in 6.0 μl of ethyl acetate. Fivemicroliters (5.0 μl) thereof was spotted to a silica gel TLC plate (TLCplate silica gel 60F₂₅₄, 20 cm×20 cm, 0.25 mm thick, Merck Company). TheTLC plate was developed with a 6:1:2 mixture of chloroform, acetic acidand ethyl acetate for about 1 hour. The solvents were then allowed toevaporate. The TLC plate was exposed overnight to an imaging plate (FujiFilm Company). Next, the imaging plate was analyzed on Image AnalyzerBAS2000 (Fuji Film Company). The presence of a spot corresponding tocompound (III) labeled with ¹⁴C were examined (Rf value 0.24 and 0.29).The results are shown in Table 14. TABLE 14 Reaction componentscomponent component component crude cell compound (II) spot of A B Cextract labeled with¹⁴C compound (III) + + + − + − + + + + + + − + + + +− + − − + + −(3) Fractionation of the Crude Cell Extract

Ammonium sulfate was added to the crude cell extract obtained in Example25(1) to amount to 45% saturation After stirring in ice-cooledconditions, the supernatant was recovered by centrifuging for 30 minutesat 12,000×g. After adding ammonium sulfate to the obtained supernatantto amount to 55% saturation and stirring in ice-cooled conditions, apellet was recovered by centrifuging for 10 minutes at 12,000×g. Thepellet was dissolved with 12.5 ml of 20 mM bistrispropane buffer(pH7.0). This solution was subjected to a PD10 column (AmershamPharmacia Company) and eluted with 20 mM of bistispropane buffer (pH7.0)to recover 17.5 ml of fractions containing proteins (hereinafterreferred to as the “45-55% ammonium sulfate fraction”).

(4) Isolation of the Present Invention Protein (A4)

The 45-55% ammonium sulfate fraction prepared in Example 25(3) wasinjected into a HiLoad26/10 Q Sepharose HP column (Amersham PharmaciaCompany). Next, after flowing 100 ml of 20 mM bistrispropane buffer(pH7.0) into the column, 20 mM bistrispropane buffer was flown with alinear gradient of NaCl (gradient of NaCl was 0.004 M/minute, range ofNaCl concentration was from 0M to 1M, flow rate was 4 ml/minute) tofraction recover 30 ml of fractions eluting at the NaCl concentration offrom 0.12M to 0.165M. Further, the recovered fractions were subjected toa PD10 column (Amersham Pharmacia Biotech Company) and eluted with 20 mMbistrispropane buffer (pH7.0) to recover the fractions containingprotein.

The recovered fractions were subjected to a PD10 column (AmershamPharmacia Biotech Company) with the elution with Buffer A (2 mMpotassium phosphate buffer containing 1.5 mM of NaCl, pH 7.0), in orderto recover the fractions containing protein. Nex the fractions wereinjected into a Bio-Scale Ceramic Hydroxyapatite Type I column CHT10-I(BioRad Company). Twenty milliliters (20 ml) of Buffer A was flown intothe column. Subsequently, Buffer A was flown with a linear gradient ofBuffer B (100 mM potassium phosphate buffer containing 0.03 mM of NaCl;the linear gradient started at 100% Buffer A to increase to 50% Buffer Bover a 100 minute period, flow rate was 2 ml/minute) to fraction recoverthe fractions eluting at a Buffer B concentration of from 4% to 6%.Further, the recovered fractions were subjected to a PD10 column(Amersham Pharmacia Biotech Company) and eluted with 0.05M potassiumphosphate buffer (pH7.0) to recover the fractions containing protein.

A similar amount of 0.05M potassium phosphate buffer (pH7.0) containing2.0M ammonium sulfate was added and mixed into the recovered fractions.The recovered fractions were then injected into a 1 ml RESOURSE PHEcolumn (Amersham Pharmacia Biotech Company). After flowing 5 ml of 0.05Mpotassium phosphate buffer (pH7.0) containing 1M ammonium sulfate, the0.05M potassium phosphate buffer (pH7.0) was flown with a lineargradient of ammonium sulfate (gradient of the ammonium sulfateconcentration was 0.1M/minute, range of NaCl concentration was 1M to 0M,flow rate was 2 ml/minute) to fraction recover the fractions eluting atan ammonium sulfate concentration of from about 0.4M to 0.5M. Theprotein contained in each of the fractions were analyzed on a 10%-20%SDS-PAGE.

Instead of the crude cell extract in the reaction solutions described inExample 25(2), the recovered fractions were added and maintained in thepresence of component A, component B, component C and compound (II)labeled with ¹⁴C, similarly to Example 25(2). The reaction solutionsafter the maintenance were TLC analyzed to examine the intensity of thespots corresponding to compound (III) labeled with ¹⁴C. Said proteinmoving to a location of about 45 kDa in the above SDS-PAGE was recoveredfrom the gel and was subjected to an amino acid sequence analysis with aprotein sequencer (Applied Biosystems Company, Procise 494HT, pulsedliquid method) to sequence the N terminus amino acid sequence. As aresult, the amino acid sequence shown in SEQ ID NO: 113 was provided.

Example 26 Obtaining the Present Invention DNA (A4)

(1) Preparation of the Chromosomal DNA of Streptomyces achromogenes IFO12735

Streptomyces achromogenes IFO 12735 cultured with shaking at 30° C. for1 day to 3 days in 50 ml of YEME medium (0.3%(w/v) yeast extract,0.5%(w/v) bacto-peptone, 0.3%(w/v) malt extract, 1.0%(w/v) glucose,34%(w/v) sucrose and 0.2%(v/v) 2.5M MgCl₂.6H₂O). The cells wererecovered. The obtained cells were suspended in YEME medium containing1.4%(w/v) glycine and 60 mM EDTA and further incubated with shaking fora day. The cells were recovered from the culture medium. After washingonce with distilled water, it was resuspended in buffer (100 mM Tris-HCl(pH8.0), 100 mM EDTA, 10 mM NaCl) at 1 ml per 200 mg of the cells. Twohundred micrograms per milliliter (200 μg/ml) of egg-white lysozyme wereadded. The cell suspension was shaken at 30° C. for a hour. Further,0.5% of SDS and 1 mg/ml of Proteinase K was added. The cell suspensionwas incubated at 55° C. for 3 hours. The cell suspension was extractedtwice with phenol.chloroform.isoamyl alcohol to recover each of theaqueous layers. Next, there was one extraction with chloroform.isoamylalcohol to recover the aqueous layer. The chromosomal DNA was obtainedby ethanol precipitating the aqueous layer.

(2) Preparation of the Chromosomal DNA Library of Streptomycesachromogenes IFO12735

Thirty-eight micrograms (38 μg) of the chromosomal DNA prepared inExample 26(1) were digested with 3.2 U of restriction enzyme Sau3A1 at37° C. for 60 minutes. The obtained digestion solution was separatedwith 1% agarose gel electrophoresis. The DNA of about 2.0 kbp wasrecovered from the gel. The DNA was purified with QIAquick GelExtraction Kit (Qiagen Company) according to the instructions attachedto said kit and was concentrated with an ethanol precipitation to obtain20 μl of the solution containing the target DNA. Eight microliters (8μl) of the DNA solution, 100 ng of plasmid vector pUC118 digested withrestriction enzyme BamHI and treated with dephosphorylation and 16 μl ofthe I solution from Ligation Kit Verb 2 (Takara Shuzo Company) weremixed and maintained for 3 hours at 16° C. E coli DH5α, were transformedutilizing the ligation solution and were spread onto LB agar mediumcontaining 50 mg/l of ampicillin to culture overnight at 37° C. Theobtained colonies were recovered from an agar medium. The plasmid wasextracted. The obtained plasmids were designated as the chromosomal DNAlibrary.

(3) Isolation of the Present Invention DNA (A4)

PCR was conducted by utilizing the chromosomal DNA prepared in Example26(2) as the template. As the primers, there was utilized the pairing ofan oligonucleotide having the nucleotide sequence shown in SEQ ID NO:114 and an oligonucleotide having the nucleotide sequence shown in SEQID NO: 57. The nucleotide sequence shown in SEQ ID NO: 114 was designedbased on the amino acid sequence shown in SEQ ID NO: 113. The ExpandHiFi PCR System (Boehringer Manheim Company) was utilized to prepare thereaction solution. The PCR reaction solution amounted to 25 μl by adding2.5 μl of the above chromosomal DNA library, the 2 primers eachamounting to 7.5 pmol, 0.2 μl of dNTP mix (a mixture of 2 mM of each ofthe 4 types of dNTP), 0.2 μl of 10× buffer (containing MgCl₂), 0.38 μlof Expand HiFi enzyme mix and distilled water. The reaction conditionsof the PCR were after maintaining 97° C. for 2 minute, repeating 10cycles of a cycle that included maintaining 97° C. for 15 seconds,followed by 65° C. for 30 seconds and followed by 72° C. for 1 minute;then conducting 15 cycles of a cycle that included maintaining 97° C.for 15 seconds, followed by 65° C. for 30 seconds and followed by 72° C.for 1 minute (wherein 20 seconds was added to the maintenance at 72° C.for each cycle); and then maintaining 72° C. for 7 minutes. After themaintenance, 2.5 μl of the reaction solution was utilized as a templatesolution for conducting PCR for a second time. As the primers, there wasutilized the pairing of an oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 115 and an oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 57. The nucleotide sequenceshown in SEQ ID NO: 1115 was designed based on the amino acid sequenceshown in SEQ ID NO: 113. Similar to the above method, the Expand HiFiPCR System (Boehringer Manheim Company) was utilized to conduct PCR. Thereaction solution after the maintenance was subjected to 2% agarose gelelectrophoresis. The gel area containing the DNA of about 800 bp wasrecovered. The DNA was purified from the recovered gel by utilizing QIAquick gel extraction kit (Qiagen Company) according to the attachedinstructions. The obtained DNA was ligated to the TA cloning vectorpCRII-TOPO (Invitrogen Company) according to the instructions attachedto said vector and was introduced into E Coli TOP10F′. The plasmid DNAwas prepared from the obtained E. coli transformant, utilizing QiagenTip20 (Qiagen Company). A sequencing reaction was conducted with Big Dyeterminator cycle sequencing FS ready reaction kit (Applied BiosystemsJapan Company) according to the instructions attached to said kit,utilizing a primers having the nucleotide sequence shown in SEQ ID NO:67 and a primer having the nucleotide sequence shown in SEQ ID NO: 68.The obtained plasmid was utilized as a template in the sequencingreaction The reaction products were analyzed with a DNA sequencer 3100(Applied Biosystems Japan Company). As a result, the nucleotide sequenceshown in nucleotides 57 to 832 of the nucleotide sequence shown in SEQID NO: 110 was provided. In the provided nucleotide sequence,nucleotides 5860 of the nucleotide sequence shown in SEQ ID NO: 110encoded amino acid 20 in the amino acid sequence shown in SEQ ID NO:113.

Next, PCR was conducted with the Expand HiFi PCR System (BoehringerManheim Company) under the above-described conditions, utilizing as atemplate the chromosomal DNA library prepared in Example 26(2). As theprimers, there was utilized a primer pairing of the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 116 and theoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 59.The amplified DNA of about 1.4 kbp was cloned into the cloning vectorpCRII-TOPO. The plasmid DNA was prepared from the obtained E. colitansformants, utilizing Qiagen Tip20 (Qiagen Company). A sequencingreaction was conducted with Big Dye terminator cycle sequencing FS readyreaction kit (Applied Biosystems Japan Company) according to theinstructions attached to said kit, utilizing a primer having thenucleotide sequence shown in SEQ ID NO: 67 and a primer having thenucleotide sequence shown in SEQ ID NO: 68. The obtained plasmid wasutilized as a template in the sequencing reaction. The reaction productswere analyzed with a DNA sequencer 3100 (Applied Biosystems JapanCompany). As a result, the nucleotide sequence shown in nucleotides 1 to58 in the nucleotide sequence shown in SEQ ID NO: 110 was provided.

The cloning of the DNA elongating downstream from the 3′ terminus of thenucleotide shown as nucleotide 832 of the nucleotide sequence shown inSEQ ID NO: 110 was conducted. Specifically, 13 μg of the chromosomal DNAof Streptomyces achromogenes IFO 12735 prepared in Example 26(1) wasdigested overnight with 200 U of restriction enzyme HincII at 37° C.After a phenol extraction, the DNA was purified by an ethanolprecipitation. The obtained DNA was used to produce 20 μl of an aqueoussolution. Four microliters (4 μl) thereof, 1.9 μl of 15 μM Genome WalkerAdaptor, 1.6 μl of 10× ligation buffer and 0.5 μl of 6 U/μl T4 ligasewere mixed and maintained overnight at 16° C. After at, there was amaintenance at 70° C. for 5 minutes and an addition of 72 μl ofdistilled water to provide a Genome Walker library. PCR was conducted byutilizing said library as a template. A PCR reaction solution amountingto 50 μl was provided by adding 1 μl of Genome Walker library and primerAP1 (provided with Universal Genome Walker Kit) and the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 117 to each amount to200 nM, adding 1 μl of dNTP mix (a mixture of 10 mM each of the 4 typesof dNTPs), 10 μl of 5×GC genomic PCR buffer, 2.2 μl of 25 mM Mg(OAc)₂,10 μl of 5M GC-Melt and 1 μl of Advantage-GC genomic polymerase mix andadding distilled water. The reaction conditions of the PCR were aftermaintaining 95° C. for 1 minute; conducting 7 cycles of a cycle thatincluded maintaining 94° C. for 10 seconds and then 72° C. for 3minutes; 36 cycles of a cycle that included maintaining 94° C. for 10seconds and then 68° C. for 3 minutes; and maintaining 68° C. for 7minutes. The reaction solution after the maintenance was diluted 50 foldwith distilled water. The PCR products were designated as the first PCRproducts and were utilized as a template to conduct another PCR. The PCRamounting 50 μl was provided by adding 1 μl of the first PCR productsand primer AP2 (provided with Universal Genome Walker Kit) and theoligonucleotide shown in SEQ ID NO: 118 to each amount to 200 nM, adding1 μl of dNTP mix (a mixture of 10 mM each of the 4 types of dNTPs), 10μl of 5×GC genomic PCR buffer, 2.2 μl of 25 mM Mg(OAc)₂, 10 μl of 5MGC-Melt and 1 μl of Advantage-CC genomic polymerase mix and addingdistilled water. The reaction conditions of the PCR were aftermaintaining 95° C. for 1 minute; conducting 5 cycles of a cycle thatincluded maintaining 94° C. for 10 seconds and then 72° C. for 3minutes; 20 cycles of a cycle that included maintaining 94° C. for 10seconds and then 68° C. for 3 minutes; and maintaining 68° C. for 7minutes. The reaction solution after the maintenance was subjected to 1%agarose gel electrophoresis. The gel area containing the DNA of about1300 bp was recovered. The DNA was purified from the recovered gel byutilizing QIA quick gel extraction kit (Qiagen Company) according to theattached instructions. The obtained DNA was ligated to cloning vectorpCRII-TOPO (Invitrogen Company) according to the instructions attachedto said vector and was introduced into E. Coli TOP10F′. The plasmid DNAwas prepared from the E. coli transformant by utilizing Qiagen Tip20(Qiagen Company). A sequencing reaction was conducted with Big Dyeterminator cycle sequencing FS ready reaction kit (Applied BiosystemsJapan Company) according to the instructions attached to said kitutilizing as primers the oligonucleotide shown in SEQ ID NO: 67 and theoligonucleotide shown in SEQ ID NO: 68. The obtained plasmid wasutilized as a template in the sequencing reaction. The reaction productswere analyzed with a DNA sequencer 3100 (Applied Biosystems JapanCompany). As a result, the nucleotide sequence shown in nucleotides 644to 1454 in the nucleotide sequence shown in SEQ ID NO: 110 was provided.As a result of connecting all of the analyzed nucleotide sequences, thenucleotide sequence shown in SEQ ID No: 110 was provided Two openreading frames (ORF) were present in said nucleotide sequence. As such,there was contained a nucleotide sequence (SEQ ID NO 109) consisting of1236 nucleotides (inclusive of the stop codon) and encoding a 411 aminoacid residue (SEQ ID NO: 108) and a nucleotide sequence (SEQ ID NO: 112)consisting of 192 nucleotides (inclusive of the stop codon) and encodinga 63 amino acid residue (SEQ ID NO: 111). The molecular weight of theprotein consisting of the amino acid sequence (SEQ ID NO: 108) encodedby the nucleotide sequence shown in SEQ ID NO: 109 was calculated to be45465 Da. Further, the amino acid sequence encoded by said nucleotidesequence contained the amino acid sequence (SEQ ID NO: 113) determinedfrom the amino acid sequencing of from the N terminus of the presentinvention protein (A4). The molecular weight of the protein consistingof the amino acid sequence (SEQ ID NO: 111) encoded by the nucleotidesequence shown in SEQ ID NO: 112 was calculated to be 6871 Da.

Example 27 The Expression of the Present Invention Protein (A4) in E.Coli

(1) Production of a Transformed E. coli Having the Present InventionDNA(A4)

PCR was conducted by utilizing as a template the chromosomal DNAprepared from Streptomyces achromogenes IFO 12735 in Example 26(1) andby utilizing Expand HiFi PCR System (Boehringer Manheim Company). As theprimers, there was utilized the pairing of an oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 119 and an oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 120 (hereinafterreferred to as “primer pairing 25”) or a pairing of an oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 119 and anoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 121hereinafter referred to as “primer pairing 26”). The PCR reactionsolution amounted to 50 μl by adding the 2 primers each amounting to 300nM, 50 ng of the above chromosomal DNA, 5.0 μl of dNTP mix (a mixture of2.0 mM of each of the 4 types of dNTP), 5.0 μl of 10× Expand HF buffer(containing MgCl₂) and 0.75 μl of Expand HiFi enzyme mix and distilledwater. The reaction conditions of the PCR were after maintaining 97° C.for 2 minutes; repeating 10 cycles of a cycle that included maintaining97° C. for 15 seconds, followed by 60° C. for 30 seconds and followed by72° C. for 1 minute; then conducting 15 cycles of a cycle that includedmaintaining 97° C. for 15 seconds, followed by 60° C. for 30 seconds andfollowed by 72° C. for 1 minute (wherein 20 seconds was added to themaintenance at 72° C. for each cycle); and then maintaining 72° C. for 7minutes. After the maintenance, the reaction solution was subjected to1% agarose gel electrophoresis. The gel area containing the DNA of about1.3 kbp was recovered from the gel which was subjected the reactionsolution utilizing primer pairing 25. The gel area containing the DNA ofabout 1.6 kbp was recovered from the gel which was subjected thereaction solution utilizing primer pairing 26. The DNA were purifiedfrom each of the recovered gels by utilizing QIA quick gel extractionkit (Qiagen Company) according to the attached instructions. Theobtained DNA were ligated to the cloning vector pCRII-TOPO (InvitrogenCompany) according to the instructions attached to said vector and wereintroduced into E. Coli TOP10F′. The plasmid DNA were prepared from theobtained B. coli transformants, utilizing Qiagen Tip20 (Qiagen Company).Next, sequencing reactions were conducted with Big Dye terminator cyclesequencing FS ready reaction kit (Applied Biosystems Japan Company)according to the instructions attached to said kit, utilizing as primersthe oligonucleotides shown in SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO;122 and SEQ ID NO: 123. The sequencing reactions utilized the obtainedplasmid DNA as the template. The reaction products were analyzed with aDNA sequencer 3100 (Applied Biosystems Japan Company). Based on theresults, the plasmid having the nucleotide sequence shown in SEQ ID NO:109 was designated as pCR646 and the plasmid having the nucleotidesequence shown in SEQ ID NO: 110 was designated as pCR646F.

Next, each of plasmids pCR646 and pCR646F was digested with restrictionenzymes NdeI and HindIII. The digestion products were subjected toagarose gel electrophoresis. The gel area containing a DNA of about 1.3kbp was cut from the gel subjected to the digestion products of pCR646.The gel area containing a DNA of about 1.6 kbp was cut from the gelsubjected to the digestion products of pCR646F. The DNA were purifiedfrom each of the recovered gels by utilizing QIA quick gel extractionkit (Qiagen Company) according to the attached instructions. Each of theobtained DNA and the plasmid pKSN2 digested with NdeI and HindIII wereligated with ligation kit Ver.1 (Takara Shuzo Company) according to theinstructions attached to said kit and introduced into E. Coli JM109. Theplasmid DNA were prepared from the obtained E. coli transformants. Thestructures thereof were analyzed. The plasmid containing the nucleotidesequence shown in SEQ ID NO: 109, in which the DNA of about 1.3 kbpencoding the present invention protein (A4) is inserted between the NdeIsite and the HindIII site of pKSN2 was designated as pKSN646. Further,the plasmid containing the nucleotide sequence shown in SEQ ID NO: 110,in which the DNA of about 1.6 kbp encoding the present invention protein(A4) is inserted between the NdeI site and the HindIII site of pKSN2 wasdesignated as pKSN646F. Each of the above plasmids of pKSN646 andpKSN646F was introduced into E. coli JM109. The obtained E. colitransformants were designated, respectively, JM109/pKSN646 andJM109/pKSN646F. Further, plasmid pKSN2 was introduced into E. coliJM109. The obtained E. coli transformant was designated as JM109/pKSN2.

(2) Expression of the Present Invention Protein (A4) in E. coli andRecovery of said Protein

E. coli JM109/pKSN646, JM109/pKSN646F and JMT109/pKSN2 are each culturedovernight at 37° C. in 10 ml of TB medium (1.2%(w/v) tryptone, 24%(w/v)yeast extract, 0.4%(w/v) glycerol, 17 mM potassium dihydrogenphosphate,72 mM dipotassium hydrogenphosphate) containing 50 μg/ml of ampicillin.A milliliter (1 ml) of the obtained culture medium is transferred to 100ml of TB medium containing 50 μg/ml of ampicillin and cultured at 26° C.When OD660 reaches about 0.5, 5-aminolevulinic acid is added to thefinal concentration of 500 μM, and the culturing is continued, Thirty(30) minutes thereafter, IPTG is added to a final concentration of 1 mM,and there is further culturing for 17 hours.

The cells are recovered from each of the culture mediums, washed with0.1M tris-HCl buffer (pH7.5) and suspended in 10 ml of the above buffercontaining 1 mM PMSF. The obtained cell suspensions are subjected 6times to a sonicator (Sonifier (Branson Sonic Power Company)) at 3minutes each under the conditions of output 3, duty cycle 30%, in orderto obtain cell lysate solutions. After centrifuging the cell lysatesolutions (1,200×g, 5 minutes) the supernatants are recovered andcentrifuged (150,000×g, 70 minutes) to recover supernatant fractions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN646 is referred to as “E. coli pKSN646 extract”, thesupernatant fraction obtained from E. coli JM109/pKSN646F is referred toas “E. coli pKSN646F extract”, and the supernatant fraction obtainedfrom E. coli JM109/pKSN2 is referred to as “E. coli pKSN2 extract”), Amicroliter (1 μl) of the above supernatant fractions is analyzed on a15% to 25% SDS-PAGE and stained with CBB. As a result, by detectingnotably more intense bands in both E. coli pKSN646 extract and E. colipKSN646F extract than the E. coli pKSN2 extract, at the electrophoresislocations corresponding to the molecular weight of 45 kDa, it can beconfirmed that the present invention protein (A4) is expressed in E.coli.

(3) Detection of the Ability to Convert Compound (I) to Compound (III)

Reaction solutions of 30 μl are prepared and maintained for 10 minutesat 30° C. The reaction solutions consist of a 0.1M potassium phosphatebuffer (pH7.0) containing 3 ppm of compound (II) labeled with ¹⁴C, 2 mMof β-NADPH (hereinafter, referred to as “component A”) (Oriental YeastCompany), 2 mg/ml of a ferredoxin derived from spinach (hereinafterreferred to as “component B”) (Sigma Company), 0.1 U/ml of ferredoxinreductase (hereinafter, referred to as “component C”) (Sigma Company)and 18 μl of the supernatant fraction recovered in Example 27(2).Further, there are prepared and maintained similarly reaction solutionshaving no addition of at least one component utilized in the compositionof the above reaction solution, selected from component A, component Band component C. Three microliters (3 μl) of 2N HCl and 90 μl of ethylacetate are added and mixed into each of the reaction solutions afterthe maintenance The resulting reaction solutions are centrifuged at8,000×g to recover 75 μl of the ethyl acetate layer. After drying theethyl acetate layers under reduced pressure, the residue is dissolved in6.0 μl of ethyl acetate. Five microliters (5.0 μl) thereof is spotted toa silica gel TLC plate (TLC plate silica gel 60F₂₅₄, 20 cm×20 cm, 0.25mm thick, Merck Company). The TLC plate is developed with a 6:1:2mixture of chloroform, acetic acid and ethyl acetate for about 1 hour.The solvents are then allowed to evaporate. The TLC plate is exposedovernight to an imaging plate (Fuji Film Company). Next, the imagingplate was analyzed on Image Analyzer BAS2000 (Fuji Film Company). Thepresence of a spot corresponding to compound (III) labeled with ¹⁴C isexamined (Rf value 0.24 and 0.29). The production of compound (III) inreaction solutions containing component A, component B, component C andE. coli pKSN646 extract, or in reaction solutions containing componentA, component B, component C and E. coli pKSN646F extract can beconfirmed.

Example 28 Sequence Identity Relating to the Present Invention Protein

The sequence identity relating to the proteins of the present inventionand the DNA of the present invention was analyzed by utilizingGENETYX-WIN Ver.5 (Software Development Company). The alignments wereproduced by conducting the homology analysis with the Lipman-Pearsonmethod (Lipman, D. J. and Pearson, W. R., Science, 227, 1435-1441,(1985)).

In regards to amino acid sequences of the present invention proteins(A1) to (A4), there were determined the sequence identities to eachother and to known proteins of the highest homology. The results areshown in Table 15. TABLE 15 present present present present knownproteins of invention invention invention invention the highest protein(A1) protein (A2) protein (A3) protein (A4) homology* present 100%  47%64% 48% 73% invention AAC25766 protein (A 1) present 47% 100%  48% 51%52% invention CAB46536 protein (A2) present 64% 48% 100%  46% 67%invention AAC25766 protein (A3) present 48% 51% 46% 100%  50% inventionCAB46536 protein (A4)*the sequence identity is shown on top and the accession number of theprovided protein in the Entrez database (provided by Center forBiotechnology Information, http://www3.ncbi.nlm.nih.gov/Entrez/) isshown on the bottom.

In regards to the nucleotide sequences of the present invention DNA (A1)having the nucleotide sequence shown in SEQ ID NO: 6, the presentinvention DNA (A2) having the nucleotide sequence shown in SEQ ID NO: 7,the present invention DNA (A3) having the nucleotide sequence shown inSEQ ID NO: 8 and the present invention DNA (A4) having the nucleotidesequence shown in SEQ ID NO: 109, there were determined the sequenceidentities to each other and to known genes of the highest homology. Theresults are shown in Table 16. TABLE 16 SEQ ID NO: 6 SEQ ID NO: 7 SEQ IDNO: 8 SEQ ID NO: 109 [present [present [present [present known genesinvention invention invention invention of the highest DNA (A1)] DNA(A2)] DNA (A3)] DNA (A4)] homology* SEQ ID NO: 6 100%  61% 74% 62% 77%[present invention AF072709 DNA (A1)] SEQ ID NO: 7 61% 100%  64% 65% 66%[present invention Y18574 DNA (A2)] SEQ ED NO: 8 74% 64% 100%  63% 75%[present invention AF072709 DNA (A3)] SEQ ID NO: 109 62% 65% 63% 100% 64% [present invention Y18574 DNA (A4)]*the sequence identity is shown on top and the accession number of theprovided gene in the Entrez database (provided by Center forBiotechnology Information, http://www3.ncbi.nlm.nih.gov/Entrez/) isshown on the bottom.

In regards to the amino acid sequences of the present invention proteins(B1) to (B4), there were determined the sequence identities to eachother and to known proteins of the highest homology. The results areshown in Table 17. TABLE 17 present present present present knownproteins of invention invention invention invention the highest protein(B1) protein (B2) protein (B3) protein (B4) homology* present 100%  45%78% 41% 76% invention AAC25765 protein (B1) present 45% 100%  40% 41%60% invention AAF71770 protein (B2) present 78% 40% 100% 40% 73%invention AAC25765 protein (B3) present 41% 41% 40% 100%  55% inventionAAA26824 protein (B4)*the sequence identity is shown on top and the accession number of theprovided protein in the Entrez database (provided by Center forBiotechnology Information, http://www3.ncbi.nlm.nih.gov/Entrez/) isshown on the bottom.

In regards to the nucleotide sequences of the present invention DNA (B1)having the nucleotide sequence shown in SEQ ID NO: 15, the presentinvention DNA (B2) having the nucleotide sequence shown in SEQ ID NO:16, the present invention DNA (B3) having the nucleotide sequence shownin SEQ ID NO: 17 and the present invention DNA (B4) having thenucleotide sequence shown in SEQ ID NO: 112, there were determined thesequence identities to each other and to known genes of the highesthomology. The results are shown in Table 18. TABLE 18 SEQ ID NO: 15 SEQID NO: 16 SEQ ID NO: 17 SEQ ID NO: 112 [present [present [present[present known genes invention invention invention invention of thehighest DNA (B1)] DNA (B2)] DNA (B3)] DNA (B4)] homology* SEQ ID NO: 15100%  60% 80% 59% 84% [present invention AF072709 DNA (B1)] SEQ ID NO:16 60% 100%  60% 59% 66% [present invention M32238 DNA (B2)] SEQ ID NO:17 80% 60% 100%  65% 79% [present invention AF072709 DNA (B3)] SEQ IDNO: 112 59% 59% 65% 100%  66% [present invention M32239 DNA (B4)]*the sequence identity is shown on top and the accession number of theprovided gene in the Entrez database (provided by Center forBiotechnology Information, http://www3.ncbi.nlm.nih.gov/Entrez/) isshown on the bottom.

Example 29 PCR Utilizing an Oligonucleotide Having a Partial NucleotideSequence of the Present Invention DNA (A) as a Primer

PCR was conducted by utilizing as a template each of: the chromosomalDNA of Streptomyces phaeochromogenes IFO 12898 prepared in Example 2;the chromosomal DNA of Saccharopolyspora taberi JCM 9383t prepared inExample 5; the chromosomal DNA of Streptomyces griseolus ATCC 11796prepared in Example 9; the chromosomal DNA of Streptomyces testaceusATCC 21469 prepared in Example 11; the chromosomal DNA of Streptomycesachromogenes IFO 12735 prepared in Example 26; and each of thechromosomal DNA of Streptomyces griseofuscus IFO 12870t, Streptomycesthermocoerulescens IFO 14273t and Streptomyces nogalater IFO 13445prepared similarly to the method described in Example 2. As the primers,the 5 pairings of primers shown in Table 19 were utilized. The predictedsize of the DNA amplified by the PCR utilizing each of the primerpairings based on the nucleotide sequence shown in SEQ ID NO: 6 is shownin Table 19. TABLE 19 primer pairing primer primer amplified DNA 14 SEQID NO: 124 SEQ ID NO: 129 about 800 bp 15 SEQ ID NO: 125 SEQ ID NO: 129about 600 bp 16 SEQ ID NO: 126 SEQ ID NO: 129 about 600 bp 17 SEQ ID NO:127 SEQ ID NO: 129 about 580 bp 18 SEQ ID NO: 128 SEQ ID NO: 129 about580 bp

The PCR reaction solution amounted to 25 μl by adding 200 nM of each ofthe 2 primers of the pairing shown in Table 19, adding 10 ng of thechromosomal DNA, 0.5 μl of dNTP mix (a mixture of 10 mM of each of the 4types of dNTP), 5 μl of 5×GC genomic PCR buffer, 1.1 μl of 25 mMMg(OAc)₂, 5 μl of 5M GC-Melt and 0.5 μl of Advantage-GC genomicpolymerase mix and adding water. The reaction conditions weremaintaining 95° C. for 1 minute; repeating 30 cycles of a cycle thatincluded maintaining 94° C. for 15 seconds, followed by 60° C. for 30seconds, and followed by 72° C. for 1 minute; and maintaining 72° C. for5 minutes. Each of the reaction solutions after the maintenance wasanalyzed with 3% agarose gel electrophoresis. The results are shown inFIG. 46 and in Table 20 and Table 21. The amplification of the predictedsize of the DNA was observed in each or all of the cases with primerpairings 14, 15, 16, 17 and 18 as well as in the cases of utilizing thechromosomal DNA prepared from any of the strains as a template. TABLE 20Reagents amplifi- primer cation Lane origin of the template chromosomalDNA pairing of DNA* 2 Streptomyces phaeochromogenes 14 + IFO 12898 3Streptomyces phaeochromogenes 15 + IFO 12898 4 Streptomycesphaeochromogenes 16 + IFO 12898 5 Streptomyces phaeochromogenes 17 + IFO12898 6 Streptomyces phaeochromogenes 18 + IFO 12898 9 Streptomycestestaceus 14 + ATCC 21469 10 Saccharopolyspora taberi 14 + JCM 9393t 11Streptomyces griseolus 14 + ATCC 11796 13 Streptomyces testaceus 15 +ATCC 21469 14 Saccharopolyspora taberi 15 + JCM 9393t 15 Streptomycesgriseolus 15 + ATCC 11796 16 Streptomyces testaceus 16 + ATCC 21469 17Saccharopolyspora taberi 16 + JCM 9393t 18 Streptomyces griseolus 16 +ATCC 11796 20 Streptomyces testaceus 17 + ATCC 21469 21Saccharopolyspora taberi 17 + JCM 9393t 22 Streptomyces griseolus 17 +ATCC 11796 23 Streptomyces testaceus 18 + ATCC 21469 24Saccharopolyspora taberi 18 + JCM 9393t 25 Streptomyces griseolus 18 +ATCC 11796*“+” represents that the predicted size of the DNA was detected and “−”represents that there was no detection.

TABLE 21 Reagents amplifi- primer cation Lane Origin of templatechromosomal DNA pairing of DNA* 28 Streptomyces griseofuscus 14 + IFO12870t 29 Streptomyces thermocoerulescens 14 + IFO 14273t 30Streptomyces achromogenes 14 − IFO 12735 31 Streptomyces nogalater 14 +IFO 13445 33 Streptomyces griseofuscus 15 + IFO 12870t 34 Streptomycesthermocoerulescens 15 + IFO 14273t 35 Streptomyces achromogenes 15 − IFO12735 36 Streptomyces nogalater 15 + IFO 13445 38 Streptomycesgriseofuscus 16 + IFO 12870t 39 Streptomyces thermocoerulescens 16 + IFO14273t 40 Streptomyces achromogenes 16 + IFO 12735 41 Streptomycesnogalater 16 + IFO 13445 43 Streptomyces griseofuscus 17 + IFO 12870t 44Streptomyces thermocoerulescens 17 + IFO 14273t 45 Streptomycesachromogenes 17 + IFO 12735 46 Streptomyces nogalater 17 + IFO 13445 48Streptomyces griseofuscus 18 − IFO 12870t 49 Streptomycesthermocoerulescens 18 + IFO 14273t 50 Streptomyces achromogenes 18 − IFO12735 51 Streptomyces nogalater 18 + IFO 13445*“+” represents that the predicted size of the DNA was detection and “−”represents that there was no detection.

Example 30 Hybridization Utilizing as a Probe a DNA Consisting of aPartial Nucleotide Sequence of the Present DNA (A) and the PresentInvention DNA (A)

(1) Preparation of a Probe

DNA consisting of a partial nucleotide sequence of the present inventionDNA (A1) or a partial nucleotide sequence of the present invention DNA(A1) was produced as a probe labeled with digoxigenin (DIG labeledprobe). PCR was conducted with PCR DIG Probe synthesis kit (RocheDiagnostics GmbH Company) according to the attached manual by utilizingas a template the chromosomal DNA of Streptomyces phaeochromogenes IFO12898 prepared in Example 3 and by utilizing as primers theoligonucleotide consisting of the nucleotide sequence shown in SEQ IDNO: 93 and the oligonucleotide consisting of the nucleotide sequenceshown in SEQ ID NO: 94. The PCR reaction solution amounted to 50 μl byadding the 2 primers each amounting to 200 nM, adding 50 ng of thechromosomal DNA, 2.5 μl of dNTP mix (a mixture of 2.0 mM of each of the4 types of dNTP), 2.5 μl of PCR DIG mix (a mixture of 2.0 mM of each ofthe 4 types of dNTP labeled with DIG), 5 μl of 10×PCR buffer and 0.75 μlof Expand HiFi enzyme mix and adding distilled water. The reactionconditions were after maintaining 95° C. for 2 minutes; repeating 10cycles of a cycle that included maintaining 95° C. for 10 seconds,followed by 60° C. for 30 seconds and followed by 72° C. for 2 minutes;then conducting 15 cycles of a cycle that included maintaining 95° C.for 10 seconds, followed by 60° C. for 30 seconds and followed by 72° C.for 2 minutes (wherein 20 seconds was added to the maintenance at 72° C.for each cycle); and then maintaining 72° C. for 7 minutes. The reactionsolution after the maintenance was subjected to 1% agarose gelelectrophoresis. As a result, amplification of a DNA of about 1.3 kb wasconfirmed. The amplified DNA was recovered to obtain a DNA labeled withdigoxigenin and having the nucleotide sequence shown in SEQ ID NO: 6.Under a similar method, PCR was conducted by utilizing as a template thechromosomal DNA of Streptomyces phaeochromogenes IFO 12898 and byutilizing as the primers the oligonucleotide consisting of thenucleotide sequence shown in SEQ ID NO: 130 and the oligonucleotideconsisting of the nucleotide sequence show in SEQ ID NO: 131. The DNAamplified by said PCR was recovered to obtain a DNA labeled withdigoxigenin and having the nucleotide sequence shown in nucleotides 57to 730 of the nucleotide sequence shown in SEQ ID NO: 6.

Under a similar method, PCR was conducted by utilizing as a template thechromosomal DNA of Saccharopolyspora taberi JCM 9393t prepared inExample 6 and by utilizing as primers the oligonucleotide consisting ofthe nucleotide sequence shown in SEQ ID NO: 61 and the oligonucleotidesequence consisting of the nucleotide sequence shown in SEQ ID NO: 62.The DNA amplified by said PCR was recovered to obtain a DNA labeled withdigoxigenin and having the nucleotide sequence shown in SEQ ID NO:7.

Further, under a similar method, PCR was conducted by utilizing as thetemplate the chromosomal DNA of Streptomyces testaceus ATCC 21469prepared in Example 11 and by utilizing as primers the oligonucleotideconsisting of the nucleotide sequence shown in SEQ ID NO: 70 and theoligonucleotide sequence consisting of the nucleotide sequence shown inSEQ ID NO: 71. The DNA amplified by said PCR was recovered to obtain aDNA labeled with digoxigenin and having the nucleotide sequence shown inSEQ ID NO: 8. Further, PCR was conducted by utilizing theabove-mentioned chromosomal DNA as the template and by utilizing as theprimers the oligonucleotide consisting of the nucleotide sequence shownin SEQ ID NO: 132 and the oligonucleotide consisting of the nucleotidesequence shown in SEQ ID NO: 133. The DNA amplified by said PCR wasrecovered to obtain a DNA labeled with digoxigenin and having thenucleotide sequence shown in nucleotides 21 to 691 of the nucleotidesequence shown in SEQ ID NO: 8.

(2) Dot-Blot Hybridization

Each of the DNA of pKSN657 prepared in Example 4 (the DNA comprising thepresent invention DNA (A1)), the DNA of pKSN923 prepared in Example 7(the DNA comprising the present invention DNA (A2)), the DNA of pKSN671prepared in Example 12 (the DNA comprising the present invention DNA(A3)), the DNA of pKSNSCA prepared in Example 14 (the DNA comprising thepresent DNA (A9)) and the DNA of pKSN11796 prepared in Example 10 (theDNA comprising the present DNA (A10)) was blotted onto a nylon membraneHybond N+ (Amersham Pharmacia Company) to amount to 100 ng and 10 ng.Ultraviolet light was directed at the obtained membranes with atransilluminator for 5 minutes.

DIG-High Prime DNA Labeling and Detection Starter Kit II (RocheDiagnostics GmbH Company) was utilized for the hybridization anddetection according to the attached manual. As the probes, each of theDNA labeled with digoxigenin and produced in Example 30(1) which weremaintained at 100° C. for 5 minutes and then quickly cooled in ice(hereinafter, referred to as “DIG labeled probe”) was utilized. Thedotted above membrane was shaken at 42° C. for 30 minutes in 2.0 ml ofDIGEasyHyb that was provided with said kit. Next, 2.0 ml of Dig EasyHyb, 5.0 μl of the DIG labeled probes and the membrane were enclosed ina plastic bag for hybridization and maintained at 42° C. for 18 hours.The membrane was recovered, was shaken twice in 2×SSC containing 0.1%SDS for 5 minutes at room temperature and was then shaken twice in0.5×SSC containing 0.1% SDS at 65° C. for 15 minutes. Subsequently, themembrane was shaken in 50 ml of washing buffer for 2 minutes, thenshaken in 50 ml of blocking solution at room temperature for 30 minutes,then shaken in 2.0 ml of antibody solution for 30 minutes, and thenshaken twice in 50 ml of washing buffer for 15 minutes. Further, aftershaking in 50 ml of detection buffer for 5 minutes, the membrane wasenclosed in a hybridization bag with 2.0 ml of Color Substrate solutionand maintained at room temperature for 18 hours. A signal was detectedin each of the cases of conducting hybridization with each of thereagents of 10 ng and 100 ng of each of pKSN657, pKSN923, pKSN671,pKSNSCA and pKSN11796.

Example 31 Obtaining the Present Invention DNA (A11)

(1) Preparation of the Chromosomal DNA of Streptomyces nogalatorIFO13445

Streptomyces nogalator IFO 13445 was cultivated with shaking at 30° C.for 3 days in 50 ml of YGY medium (0.5%(w/v) yeast extract, 0.5%(w/v)tryptone, 0.1%(w/v) glucose and 0.1%(w/v) K₂BPO4 pH7.0). The cells wererecovered. The obtained cells were suspended in YGY medium containing1.4%(w/v) glycine and 60 mM EDTA and further incubated with shaking fora day. The cells were recovered from the culture medium. After washingonce with distilled water, it was suspended in 3.5 ml of Buffer B1 (50mM Tris-HCl (pH8.0), 50 mM EDTA, 0.5% of Tween-20 and 0.5% TritonX-100). Eighty microliters (80 μl) of a 100 μg/ml lysozyme solution and100 μl of Qiagen Protease (600 mAU/ml, Qiagen Company) were added to thesuspension and maintained at 37° C. for a hour. Next, 1.2 ml of BufferB2 (3M guanidine HCl and 20% tween-20) was added, mixed and maintainedat 50° C. for 30 minutes. The obtained cell lysate solution added to aQiagen genomic chip 100G (Qiagen Company) equalized in Buffer QBT (750mM NaCl, 50 mM MOPS (pH7.0), 15% isopropanol and 0.15% Triton X-100).Next, after the chip was washed twice with 7.5 ml of Buffer QC (50 mMMOPS (pH7.0) and 15% isopropanol), the DNA was eluted by flowing 5 ml ofBuffer QF (1.25M NaCl, 50 mM Tris HCl (pH8.5), 15% isopropanol). Threeand five-tenths milliliters (3.5 ml) of isopropanol was mixed into theobtained DNA solution to precipitate and recover the chromosomal DNA.After washing with 70% ethanol, the recovered chromosomal DNA wasdissolved in 1 ml of TB buffer.

(2) Isolation of DNA Having a Partial Nucleotide Sequence of the PresentInvention DNA (A11)

PCR was conducted by utilizing as the template the chromosomal DNAprepared in Example 31(1) and by utilizing primer pairing 14, inaccordance with the method described in Example 29. The amplified DNAwas ligated to cloning vector pCRII-TOPO (Invitrogen Company) accordingto the instructions attached to said vector and was then introduced intoE. Coli TOP10F′. The plasmid DNA was prepared from the obtained E. colitransformant, utilizing Qiagen Tip20 (Qiagen Company). A sequencingreaction was conducted with Dye terminator cycle sequencing FS readyreaction kit (Applied Biosystems Japan Company) according to theinstructions attached to said kit, utilizing a primer having thenucleotide sequence shown in SEQ ID NO: 57 and a primer having thenucleotide sequence shown in SEQ ID NO: 59. The sequence reactionutilized the obtained plasmid as a template. The reaction products wereanalyzed with a DNA sequencer 3100 (Applied Biosystems Japan Company) Asa result, the nucleotide sequence shown in nucleotides 316 to 1048 ofthe nucleotide sequence shown in SEQ ID NO: 139 was provided.

Further, the chromosomal DNA prepared in Example 31(1) was digested withrestriction enzyme PvuII. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 161 and primer AP1 (Universal Genome WalkerKit (Clontech Company)). Next, PCR was conducted under the conditionsdecribed in Example 26(3), by utilizing the first PCR products as thetemplate and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 162 and primer AP2 (Universal Genome WalkerKit (Clontech Company)). The nucleotide sequence of the obtained DNA wasanalyzed. The nucleotide sequence shown in nucleotides 1 to 330 of thenucleotide sequence shown in SEQ ID NO: 144 was provided.

Further, the chromosomal DNA prepared in Example 31(1) was digested withrestriction enzyme HincII. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 163 and primer AP1 (Universal Genome WalkerKit (Clontech Company)). Next, PCR was conducted under the conditionsdescribed in Example 26(3), by utilizing the first PCR products as thetemplate and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 164 and primer AP2 (Universal Genome WalkerKit (Clontech Company)). The nucleotide sequence of the obtained DNA wasanalyzed. The nucleotide sequence shown in nucleotides 983 to 1449 ofthe nucleotide sequence shown in SEQ ID NO: 144 was provided.

(3) Sequence Analysis of the Present Invention DNA (A11)

The nucleotide sequence shown in SEQ ID NO: 144 was obtained byconnecting the nucleotide sequences provided by the DNA obtained inExample 31(2). Two open reading frames (ORF) were present. As such,there was contained a nucleotide sequence (SEQ ID NO: 139) consisting of1230 nucleotides (inclusive of the stop codon) and encoding a 409 aminoacid residue (SEQ ID NO: 159) and a nucleotide sequence (SEQ ID NO: 154)consisting of 207 nucleotides (inclusive of the stop codon) and encodinga 68 amino acid residue (SEQ ID NO: 149). The molecular weight of theprotein consisting of the amino acid sequence (SEQ ID NO: 159) encodedby the nucleotide sequence shown in SEQ ID NO: 139 was calculated to be45177 Da. Further, the molecular weight of the protein consisting of theamino acid sequence (SEQ ID NO: 149) encoded by the nucleotide sequenceshown in SEQ ID NO: 154 was calculated to be 7147 Da.

Example 32 Expression of the Present Invention Protein (A11) in E. Coli

(1) Production of a Transformed E. coli Having the Present Invention DNA(A11)

PCR was conducted by utilizing as a template the chromosomal DNAprepared from Streptomyces nogalator IFO13445 in Example 31(1) and byutilizing Expand HiFi PCR System (Boehringer Manheim Company). As theprimers, there was utilized the pairing of an oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 165 and an oligonucleotidehaving the nucleotide sequence shown in SEQ MD NO: 166. The reactionsolution composition and the maintenance were similar to the conditionsdescribed in Example 27(1). The reaction solution after the maintenancewas subjected to 1% agarose gel electrophoresis. The gel area containingthe DNA of about 1.5 kbp was recovered. The DNA was purified from therecovered gel by utilizing QIA quick gel extraction kit (Qiagen Company)according to the attached instructions. The obtained DNA was ligated tothe cloning vector pCR1-TOPO (Invitrogen Company) according to theinstructions attached to said vector and was introduced into E. ColiTOP10F′. The plasmid DNA was prepared from the obtained E. colitransformants, utilizing Qiagen Tip20 (Qiagen Company). Sequencingreactions were conducted with Dye terminator cycle sequencing FS readyreaction kit (Applied Biosystems Japan Company) according to theinstructions attached to said kit, utilizing as primers theoligonucleotides having the nucleotide sequences shown in, respectively,SEQ ID NOs: 57, 59, and 186. The sequencing reactions utilized theobtained plasmid DNA as the template The reaction products were analyzedwith a DNA sequencer 3100 (Applied Biosystems Japan Company). Based onthe results, the plasmid having the nucleotide sequence shown in SEQ IDNO: 144 was designated as pCR849AF.

Next, pCR849AF was digested with restriction enzymes NdeI and HindIII.The digestion products were subjected to agarose gel electrophoresis.The gel area containing a DNA of about 1.5 kbp was cut from the gel. TheDNA was purified from the recovered gels by utilizing QIA quick gelextraction kit (Qiagen Company) according to the attached instructions.The obtained DNA and the plasmid pKSN2 digested with NdeI and HindIIIwere ligated with ligation kit Ver.2 (Takara Shuzo Company) according tothe instructions attached to said kit and introduced into E. Coli JM109.The plasmid DNA were prepared from the obtained E. coli tansformants.The structures thereof were analyzed. The plasmid containing thenucleotide sequence shown in SEQ ID NO: 144, in which the DNA of about1.5 kbp encoding the present invention protein (A11) is inserted betweenthe NdeI site and the HindIII site of pKSN2 was designated as pKSN849AF.Plasmid pKSN849AF was introduced into E. coli JM109. The obtained E.coli transformant was designated JM109/pKSN849AF Further, plasmid pKSN2was introduced into E. coli JM109. The obtained E. coli transformant wasdesignated as JM109/pKSN2.

(2) Expression of the Present Invention Protein (A11) in E. coli andRecovery of Said Protein

Similarly to Example 4(2), each of E. coli JM109/pKSN849AF andJM109/pKSN2 was cultured. The cells were recovered. Cell lysatesolutions were prepared. Under the method described in Example 4(2),supernatant fractions were prepared from the cell lysate solutions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN849AF is referred to as “E. coli pKSN849AF extract” and thesupernatant fraction obtained from JM109/pKSN2 is referred to as “E.coli pKSN2 extract”).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Reaction solutions of 30 μl were prepared and maintained for 10 minutesat 30° C. The reaction solutions consisted of a 0.1M potassium phosphatebuffer (pH7.0) containing 3 ppm of compound (II) labeled with ¹⁴C, 2 mMof β-NADPH (hereinafter, referred to as “component A”) (Oriental YeastCompany), 2 mg/ml of a ferredoxin derived from spinach (hereinafterreferred to as “component B”) (Sigma Company), 0.1 U/ml of ferredoxinreductase (hereinafter, referred to as “component C”) (Sigma Company)and 23 μl of the supernatant fraction recovered in Example 32(2).Similarly to Example 4(3), the reaction solutions after the maintenancewere extracted with ethyl acetate and the extracted layers were TLCanalyzed. After developing the TLC plate, the presence of a spot thereoncorresponding to compound (III) labeled with ¹⁴C were examined (Rf value0.24 and 0.29). A spot corresponding to compound (III) was detected fromthe reaction solution containing E. coli pKSN849AF extract. In contrast,such a spot was not detected from the reaction solution containing E.coli pKSN2 extract.

Example 33 Obtaining the Present Invention DNA (A12)

(1) Preparation of the Chromosomal DNA of Streptomyces tsusimaensis IFO13782

Under the method described in Example 31(1), the chromosomal DNA ofStreptomyces tsusimaensis IFO 13782 was prepared.

(2) Isolation of DNA Having a Partial Nucleotide Sequence of the PresentInvention DNA (A12)

PCR was conducted by utilizing as the template the chromosomal DNA ofStreptomyces tsusimaensis IFO 13782 prepared in Example 33(1) and byutilizing primer pairing 14, in accordance with the method described inExample 29. Similarly to Example 31(2), the amplified DNA was cloned tocloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequencethereof was analyzed. As a result, the nucleotide sequence shown innucleotides 364 to 1096 of the nucleotide sequence shown in SEQ ID NO:140 was provided.

Further, the chromosomal DNA prepared in Example 33(1) was digested withrestriction enzyme SmaI. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the fist PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 167 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 168 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1 to 392 of the nucleotide sequence shownin SEQ ID NO: 145 was provided.

Further, the chromosomal DNA prepared in Example 33(1) was digested withrestriction enzyme PvuII. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 169 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 170 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1048 to 1480 of the nucleotide sequenceshown in SEQ ID NO: 145 was provided.

(3) Sequence Analysis of the Present Invention DNA (A12)

The nucleotide sequence shown in SEQ ID NO: 145 was obtained byconnecting the nucleotide sequences provided by the DNA obtained inExample 33(2). Two open reading frames (ORF) were present in saidnucleotide sequence. As such, there was contained a nucleotide sequence(SEQ ID NO: 140) consisting of 1278 nucleotides (inclusive of the stopcodon) and encoding a 425 amino acid residue (SEQ ID NO: 160) and anucleotide sequence (SEQ ID NO: 155) consisting of 198 nucleotides(inclusive of the stop codon) and encoding a 65 amino acid residue (SEQID NO: 150). The molecular weight of te protein consisting of the aminoacid sequence (SEQ ID NO: 160) encoded by the nucleotide sequence shownin SEQ ID NO: 140 was calculated to be 46549 Da Further, the molecularweight of the protein consisting of the amino acid sequence (SEQ ID NO;150) encoded by the nucleotide sequence shown in SEQ ID NO: 155 wascalculated to be 6510 Da.

Example 34 Expression of the Present Invention DNA (A12) in E. Coli

(1) Production of a Transformed E. coli Having the Present Invention DNA(A12)

PCR was conducted similarly to Example 32(1), other than utilizing as atemplate the chromosomal DNA prepared from Streptomyces tsusimaensis IFO13782 in Example 33(1) and utilizing as the primers the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 171 and anoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 172.Similarly to Example 32(1), the DNA was purified from the reactionsolution of PCR and cloned into the cloning vector pCRII-TOPO(Invitrogen Company). The nucleotide sequence of the obtained plasmidDNA was analyzed with oligonucleotides having the nucleotide sequencesshown, respectively, in SEQ ID NOs: 57, 59, 171, 172 and 187. Based onthe obtained results, the plasmid having the nucleotide sequence shownin SEQ ID NO: 145 was designated as pCR1618F. Similarly to Example32(1), pCR1618F was digested with restriction enzymes NdeI and HindIII.A DNA of about 1.5 kbp was purified from the digestion products. Theobtained DNA and the plasmid pKSN2 digested with NdeI and HindIII wereligated to obtain a plasmid containing the nucleotide sequence shown inSEQ ID NO: 145, in which the DNA encoding the present invention protein(A12) is inserted between the NdeI site and the HindIII site of pKSN2(hereinafter referred to as “pKSN1618F”). Said plasmid was introducedinto E. Coli JM109. The obtained E. coli transformant was designatedJM109/pKSN1618F.

(2) Expression of the Present Invention Protein (A12) in E. coli andRecovery of said Protein

Similarly to Example 4(2), each of E. coli JM109/pKSN1618F andJM109/pKSN2 was cultured. The cells were recovered. Cell lysatesolutions were prepared. Under the method described in Example 4(2),supernatant fractions were prepared from the cell lysate solutions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN1618F is referred to as “E. coli pKSN1618F extract” and thesupernatant fraction obtained from E. coli JM109/pKSN2 is referred to as“E. coli pKSN2 extract”).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Reaction solutions of 30 μl were prepared and maintained for 10 minutesat 30° C. Other than utilizing the supernatant fractions recovered inExample 34(2) (E. coli pKSN1618F extract or E. coli pKSN2 extract), thereaction solutions were prepared similarly to Example 32(3). Thereaction solutions after the maintenance were extracted with ethylacetate and the extracted layers were TLC analyzed. After developing theTLC plate, the presence of a spot thereon corresponding to compound(III) labeled with ¹⁴C were examined (Rf value 0.24 and 0.29). A spotcorresponding to compound (III) was detected from the reaction solutioncontaining E. coli pKSN161SF extract. In contrast, such a spot was notdetected from the reaction solution containing E. coli pKSN2 extract.

Example 35 Obtaining the Present Invention DNA (A13)

(1) Preparation of the Chromosomal DNA of Streptomyces thermocoerulescesIFO 14273t

Under the method described in Example 31(1), the chromosomal DNA ofStreptomyces thermocoerulesces IFO 14273t was prepared.

(2) Isolation of DNA Having a Partial Nucleotide Sequence of the PresentInvention DNA (A13)

PCR was conducted by utilizing as the template the chromosomal DNA ofStreptomyces thermocoerulesces IFO 14273t prepared in Example 35(1) andby utilizing primer pairing 14, in accordance with the method describedin Example 29. Similarly to Example 31(2), the amplified DNA was clonedto cloning vector pCRII-TOPO (Invitrogen Company). The nucleotidesequence thereof was analyzed. As a result, the nucleotide sequenceshown in nucleotides 295 to 1027 of the nucleotide sequence shown in SEQID NO: 141 was provided.

Further, the chromosomal DNA prepared in Example 35(1) was digested withrestriction enzyme HincII. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 173 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 174 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1 to 370 of the nucleotide sequence shownin SEQ ID NO: 146 was provided.

Further, the chromosomal DNA prepared in Example 35(1) was digested withrestriction enzyme SmaI. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 175 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 176 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 960 to 1473 of the nucleotide sequenceshown in SEQ ID NO: 146 was provided.

(3) Sequence Analysis of the Present Invention DNA (A13)

The nucleotide sequence shown in SEQ ID NO: 146 was obtained byconnecting the nucleotide sequences provided by the DNA obtained inExample 35(2). Two open reading frames (ORF) were present in saidnucleotide sequence. As such, there was contained a nucleotide sequence(SEQ ID NO: 141) consisting of 1209 nucleotides (inclusive of the stopcodon) and encoding a 402 amino acid residue (SEQ ID NO: 136) and anucleotide sequence (SEQ ID NO: 156) consisting of 252 nucleotides(inclusive of the stop codon) and encoding a 83 amino acid residue (SEQID NO: 151). The molecular weight of the protein consisting of the aminoacid sequence (SEQ ID NO: 136) encoded by the nucleotide sequence shownin SEQ ID NO: 141 was calculated to be 44629 Da Further, the molecularweight of the protein consisting of the amino acid sequence (SEQ ID NO:151) encoded by the nucleotide sequence shown in SEQ ID NO: 156 wascalculated to be 8635 Da.

Example 36 Expression of the Present Invention DNA (A13) in E. Coli

(1) Production of a Transformed E. coli Having the Present Invention DNA(A13)

PCR was conducted similarly to Example 32(1), other than utilizing as atemplate the chromosomal DNA prepared from Streptomycesthermocoerulesces IFO 14273t in Example 35(1) and utilizing as theprimers the oligonucleotide having the nucleotide sequence shown in SEQID NO: 177 and an oligonucleotide having the nucleotide sequence shownin SEQ ID NO: 178. Similarly to Example 32(1), the DNA was purified fromthe reaction solution of PCR and cloned into the cloning vectorpCRII-TOPO (Invitrogen Company). The nucleotide sequence of the obtainedplasmid DNA was analyzed with oligonucleotides having nucleotidesequences shown, respectively, in SEQ ID NOs: 57, 59, 173, 175 and 188.Based on the obtained results, the plasmid having the nucleotidesequence shown in SEQ ID NO: 146 was designated as pCR474F. Similarly toExample 32(1), pCR474F was digested with restriction enzymes NdeI andHindIII. A DNA of about 1.5 kbp was purified from the digestionproducts. The obtained DNA and the plasmid pKSN2 digested with NdeI andHinds were ligated to obtain a plasmid containing the nucleotidesequence shown in SEQ ID NO: 146, in which the DNA encoding the presentinvention protein (A13) is inserted between the NdeI site and theHindIII site of pKSN2 (hereinafter referred to as “pKSN474F”). Saidplasmid was introduced into E. Coli JM109. The obtained E. colitransformant was designated JM109/pKSN474F.

(2) Expression of the Present Invention Protein (A13) in E. coli andRecovery of Said Protein

Similarly to Example 4(2), each of E. coli JM109pKSN474F and JM109/pKSN2was cultured. The cells were recovered. Cell lysate solutions wereprepared, Under the method described in Example 4(2), supernatantfractions were prepared from the cell lysate solutions (hereinafter, thesupernatant fraction obtained from E. coli JM109/pKSN474F is referred toas “E. coli pKSN474F extract” and the supernatant fraction obtained fromJM109/pKSN2 is referred to as “E. coli pKSN2 extract”).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Reaction solutions of 30 μl were prepared and maintained for 10 minutesat 30° C. Other than utilizing the supernatant fractions recovered inExample 36(2) (E. coli pKSN474F extract or E coli pKSN2 extract), thereaction solutions were prepared similarly to Example 32(3). Thereaction solutions after the maintenance were extracted with ethylacetate and the extracted layers were TLC analyzed. After developing theTLC plate, the presence of a spot thereon corresponding to compound(III) labeled with ¹⁴C were examined (Rf value 0.24 and 0.29). A spotcorresponding to compound (III) was detected from the reaction solutioncontaining E. coli pKSN474F extract. In contrast, such a spot was notdetected from the reaction solution containing E. coli pKSN2 extract.

Example 37 Obtaining the Present Invention DNA (A14)

(1) Preparation of the Chromosomal DNA of Streptomyces thermocoerulescesIFO 14273t

Under the method described in Example 31(1), the chromosomal DNA ofStreptomyces glomerochromogenes IFO 13673t was prepared.

(2) Isolation of DNA Having a Partial Nucleotide Sequence of the PresentInvention DNA (A13)

PCR was conducted by utilizing as the template the chromosomal DNA ofStreptomyces glomerochromogenes IFO 13673t prepared in Example 37(1) andby utilizing primer pairing 14, in accordance with the method describedin Example 29. Similarly to Example 31(2), the amplified DNA was clonedto cloning vector pCRII-TOPO (Invitrogen Company). The nucleotidesequence thereof was analyzed. As a result, the nucleotide sequenceshown in nucleotides 316 to 1048 of the nucleotide sequence shown in SEQID NO: 142 was provided.

Further, the chromosomal DNA prepared in Example 37(1) was digested withrestriction enzyme SmaI. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 179 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 180 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1 to 330 of the nucleotide sequence shownin SEQ ID NO: 147 was provided.

Further, the chromosomal DNA prepared in Example 37(1) was digested withrestriction enzyme HincII. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 181 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 182 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 982 to 1449 of the nucleotide sequenceshown in SEQ ID NO: 147 was provided.

(3) Sequence Analysis of the Present Invention DNA (A14)

The nucleotide sequence shown in SEQ ID NO: 147 was obtained byconnecting the nucleotide sequences provided by the DNA obtained inExample 37(2). Two open reading frames (ORF) were present in saidnucleotide sequence. As such, there was contained a nucleotide sequence(SEQ ID NO: 142) consisting of 1230 nucleotides (inclusive of the stopcodon) and encoding a 409 amino acid residue (SEQ ID NO: 137) and anucleotide sequence (SEQ ID NO: 157) consisting of 207 nucleotides(inclusive of the stop codon) and encoding a 68 amino acid residue (SEQID NO: 152). The molecular weight of the protein consisting of the aminoacid sequence (SEQ ID NO: 137) encoded by the nucleotide sequence shownin SEQ ID NO: 142 was calculated to be 45089 Da. Further, the molecularweight of the protein consisting of the amino acid sequence (SEQ ID NO:152) encoded by the nucleotide sequence shown in SEQ ID NO: 157 wascalculated to be 7174 Da.

Example 38 Expression of the Present Invention DNA (A14) in E. Coli

(I) Production of a Transformed E. coli Having the Present Invention DNA(A14)

PCR was conducted similarly to Example 32(1), other than utilizing as atemplate the chromosomal DNA of Streptomyces glomerochromogenes IFO13673t prepared in Example 37(1) and utilizing as the primers theoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 183and an oligonucleotide having the nucleotide sequence shown in SEQ IDNO: 184. Similarly to Example 32(1), the DNA was purified from the PCRreaction solution and cloned into cloning vector pCRII-TOPO (InvitrogenCompany). The nucleotide sequence of the obtained plasmid DNA wasanalyzed with oligonucleotides having nucleotide sequences shown,respectively, in SEQ ID NOs: 57, 59 and 189. Based on the obtainedresults, the plasmid having the nucleotide sequence shown in SEQ ID NO:147 was designated as pCR1491AF. Similarly to Example 32(1), pCR1491AFwas digested with restriction enzymes NdeI and HindIII. A DNA of about1.5 kbp was purified from the digestion products. The obtained DNA andthe plasmid pKSN2 digested with NdeI and HindIII were ligated to obtaina plasmid containing the nucleotide sequence shown in SEQ ID NO: 147, inwhich the DNA encoding the present invention protein (A14) is insertedbetween the NdeI site and the HindIII site of pKSN2 (hereinafterreferred to as “pKSN1491AF”). Said plasmid was introduced into E. ColiJM109. The obtained E. coli transformant was designatedJM109/pKSN1491AF.

(2) Expression of the Present Invention Protein (A14) in E. coli andRecovery of Said Protein

Similarly to Example 4(2), each of E. coli JM109/pKSN1491AF andJM109/pKSN2 was cultured. The cells were recovered. Cell lysatesolutions were prepared. Under the method described in Example 4(2),supernatant fractions were prepared from the cell lysate solutions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN1491AF is referred to as “E. coli pKSN1491AF extract” and thesupernatant fraction obtained from JM109/pKSN2 is referred to as “E.coli pKSN2 extract”).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Reaction solutions of 30 μl were prepared and maintained for 10 minutesat 30° C. Other than utilizing the supernatant fractions recovered inExample 38(2) (E. coli pKSN1491AF extract or E. coli pKSN2 extract), thereaction solutions were prepared similarly to Example 32(3). Thereaction solutions after the maintenance were extracted with ethylacetate and the extracted layers were TLC analyzed. After developing theTLC plate, the presence of a spot thereon corresponding to compound(III) labeled with ¹⁴C were examined (Rf value 0.24 and 0.29). A spotcorresponding to compound (III) was detected from the reaction solutioncontaining E. coli pKSN1491AF extract. In contrast, such a spot was notdetected from the reaction solution containing E. coli pKSN2 extract.

Example 39 Obtaining the Present Invention DNA (A15)

(1) Preparation of the Chromosomal DNA of Streptomyces olivochromogenesIFO 1244

Under the method described in Example 31(1), the chromosomal DNA ofStreptomyces olivochromogenes IFO 12444 was prepared.

(2) Isolation of DNA Having a Partial Nucleotide Sequence of the PresentInvention DNA (A15)

PCR was conducted by utilizing as the template the chromosomal DNA ofStreptomyces olivochromogenes IFO 12444 prepared in Example 39(1) and byutilizing primer pairing 14, in accordance with the method described inExample 29. Similarly to Example 31(2), the amplified DNA was cloned tocloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequencethereof was analyzed. As a result, the nucleotide sequence shown innucleotides 316 to 1048 of the nucleotide sequence shown in SEQ ID NO:143 was provided.

Further, the chromosomal DNA prepared in Example 37(1) was digested withrestriction enzyme SmaI. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained DNA as thetemplate and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 179 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 180 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1 to 330 of the nucleotide sequence shownin SEQ ID NO: 148 was provided.

Further, the chromosomal DNA prepared in Example 39(1) was digested withrestriction enzyme SmaI. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 181 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 182 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 982 to 1449 of the nucleotide sequenceshown in SEQ ID NO: 148 was provided.

(3) Sequence Analysis of the Present Invention DNA (A15)

The nucleotide sequence shown in SEQ ID NO: 148 was obtained byconnecting the nucleotide sequences provided by the DNA obtained inExample 39(2). Two open reading frames (ORF) were present in saidnucleotide sequence. As such, there was contained a nucleotide sequence(SEQ ID NO: 143) consisting of 1230 nucleotides (inclusive of the stopcodon) and encoding a 409 amino acid residue (SEQ ID NO: 138) and anucleotide sequence (SEQ ID NO: 158) consisting of 207 nucleotides(inclusive of the stop codon) and encoding a 68 amino acid residue (SEQID NO: 153). The molecular weight of the protein consisting of the aminoacid sequence (SEQ ID NO: 138) encoded by the nucleotide sequence shownin SEQ ID NO: 143 was calculated to be 45116 Da. Further, the molecularweight of the protein consisting of the amino acid sequence (SEQ ID NO:153) encoded by the nucleotide sequence shown in SEQ ID NO: 158 wascalculated to be 7179 Da.

Example 40 Expression of the Present Invention DNA (A15) in E. Coli

(1) Production of a Transformed E. coli Having the Present Invention DNA(A15)

PCR was conducted similarly to Example 32(1), other than utilizing as atemplate the chromosomal DNA of Streptomyces olivochromogenes IFO 12444prepared in Example 39(1) and utilizing as the primers theoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 184and an oligonucleotide having the nucleotide sequence shown in SEQ IDNO: 185 Similarly to Example 32(1), the DNA was purified from the PCRreaction solution and cloned into cloning vector pCRII-TOPO (InvitrogenCompany). The nucleotide sequence of the obtained plasmid DNA wasanalyzed with oligonucleotides having nucleotide sequences shown,respectively, in SEQ ID NOs: 57, 59 and 189. Based on the obtainedresults, the plasmid having the nucleotide sequence shown in SEQ ID NO:148 was designated as pCR1555AF. Similarly to Example 32(1), pCR155AFwas digested with restriction enzymes NdeI and HindIII. A DNA of about1.5 kbp was purified from the digestion products. The obtained DNA andthe plasmid pKSN2 digested with NdeI and HindIII were ligated to obtaina plasmid containing the nucleotide sequence shown in SEQ ID NO: 148, inwhich the DNA encoding the present invention protein (A15) is insertedbetween the NdeI site and the HindIII site of pKSN2 (hereinafterreferred to as “pKSN1555AF”). Said plasmid was introduced into E. ColiJM109. The obtained E. coli transformant was designatedJM109/pKSN1555AF.

(2) Expression of the Present Invention Protein (A15) in E. coli andRecovery of Said Protein

Similarly to Example 4(2), each of E. coli JM109/pKSN1555AF andJM109/pKSN2 was cultured. The cells were recovered. Cell lysatesolutions were prepared. Under the method described in Example 4(2),supernatant fractions were prepared from the cell lysate solutions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN1555AF is referred to as “E. coli pKSN1555AF extract” and thesupernatant fraction obtained from JM109/pKSN2 is referred to as “E.coli pKSN2 extract”).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Reaction solutions of 30 μl were prepared and maintained for 10 minutesat 30° C. Other than utilizing the supernatant fractions recovered inExample 40(2) (E. coli pKSN1555AF extract or E. coli pKSN2 extract), thereaction solutions were prepared similarly to Example 32(3). Thereaction solutions after the maintenance were extracted with ethylacetate and the extracted layers were TLC analyzed. After developing theTLC plate, the presence of a spot thereon corresponding to compound(III) labeled with ¹⁴C were examined (Rf value 0.24 and 0.29). A spotcorresponding to compound (III) was detected from the reaction solutioncontaining E. coli pKSN1555AF extract. In contrast, such a spot was notdetected from the reaction solution containing E. coli pKSN2 extract.

Example 41 Metabolism of Compounds by the Present Invention Protein (A1)

(1) Preparation of Plastid Fractions

A hundred grams (100 g) of Radish greens seeds (Takii Seed) were sawedinto a dampened paper laboratory wipe in a tray, cultivated at 25° C.for 6 days in the dark and then cultivated for 4 hours under afluorescent lamp. Thirty grams (30 g) of the newly greened cotyledonswere ground with a Nissei AM-8 homoginizer (Nihonseiki Seisakusho;18,000 to 20,000 rpm, 4° C., 5 seconds) in disruption buffer (1 mMmagnesium chloride, 20 mMN-tris(hydroxymethyl)methyl-2-aminoethanesulfonate, 10 mMN-2-hydroxyethylpiperidine-N′-2-ethanesulfonate, 0.5 mM EDTA, 5 mMcysteine, 0.5M sucrose; pH7.7). The obtained cell lysate solution waspassed trough 4 layers of nylon gause. The obtained solution wascentrifuged (13,170×g, 4° C., 1 minute). The obtained residue fractionswere suspended with 60 ml of disruption buffer and centrifuged (2,640×g,4° C., 2 minutes). The residue fractions were resuspended in 10 ml ofdisruption buffer, were layered with the high density buffer (1 mMmagnesium chloride, 20 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate, 30 mMN-2-hydroxyethylpiperidine-N′-2-ethanesulfonate, 0.5 mM EDTA, 5 mMcysteine, 0.6M sucrose; pH7.7) in a centrifuge tube, and werecentrifuged (675×g, 4° C., 15 minutes). The residues were suspended in 3ml of suspension buffer (1 mM magnesium chloride, 20 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate, 30 mMN-2-hydroxyethylpiperidine-N′-2-ethanesulfonate, 0.5 mM EDTA; pH7.7) andwere designated as a plastid fraction.

(2) Metabolism of Compound (XII) by the Present Invention Protein (A1)

There was prepared 100 μl of a reaction solution of 50 mM potassiumphosphate buffer (pH7.0) containing 5 ppm of compound (XII), 3 mM ofβ-NADPH (hereinafter, referred to as “component A”) (Oriental YeastCompany), 1 mg/ml of a ferredoxin derived from spinach (hereinafterreferred to as “component B”) (Sigma Company), 0.15 U/ml of ferredoxinreductase (hereinafter, referred to as “component C”) (Sigma Company)and 20 μl of the supernatant fraction recovered in Example 4(2). Thereaction solution was maintained at 30° C. for 10 minutes. Further,there was prepared and maintained similarly 100 μl of a reactionsolution of a 50 mM potassium phosphate buffer (pH 7.0) having noaddition of at least one component utilized in the composition of theabove reaction solution, selected from component A, component B,component C and the supernatant fraction prepared in Example 4(2). Tenmicroliters (10 μl) of 2N HCl and 500 μl of ethyl acetate were added andmixed into each of the reaction solutions after the maintenance. Theresulting reaction solutions were centrifuged at 8,000×g to recover 490μl of the ethyl acetate layer. After drying the ethyl acetate layersunder reduced pressure, the residue was dissolved in 100 μl of 50 mM ofpotassium phosphate buffer (pH7.0). Forty microliters (40 μl) of thefraction solutions (hereinafter, the fraction solution derived from thereaction solution containing component A, component B, component C and20 μl of supernatant fraction recovered in Example 4(2) is referred toas “(XII) metabolism solution (A1)”; further, the fraction solutionderived from the reaction solution containing no component A, nocomponent B, no component C and no supernatant fraction recovered inExample 4(2) is referred to as “(XII) control solution (A1)”) wereanalyzed on a HPLC. Compared to the concentration of compound (XII)detected from (XII) control solution (A1), the concentration of compound(XII) detected from (XII) metabolism solution (A1) was lower. Further apeak, which was not detected from the (XII) control solution (A1), wasdetected from the (XII) metabolism solution (A1). Mass spectrometry wasconducted for the compound contained in such a peak. The mass of thecompound contained in such a peak was 14 smaller than the mass ofcompound (XII).

Twenty microliters (20 μl) of a 32-fold dilution of the above (XII)metabolism solution (A1) and 60 μl of the plastid fraction prepared inExample 41(1) were mixed. In darkened conditions, 20 μl of substratesolution (10 mM adenosine triphosphate, 5 mM aminolevulinic acid, 4 mMglutathion reductase and 0.6 mM NAD⁺; pH6.5; hereinafter, such asubstrate solution is referred to as “PPO substrate solution”) wereadded and maintained at 30° C. for 1.5 hours. Further, instead of said20 μl of the 32-fold dilution of (XII) metabolism solution (A1), areaction solution to which 20 μl of the 32-fold dilution of (XII)control solution (A1) was added was prepared, and the PPO substratesolution was added and maintained similarly. Three hundred (300 μl) of adimethylsulfoxide-methanol mixture (dimethylsulfoxide: methanol=7:3) wasadded to each of the reaction solutions after the maintenance andcentrifuged (8000×g, 4° C., 10 minutes). The supernatants were recoveredand were subjected to reverse phase HPC analysis under the analysisconditions below to measure the amount of PPIX. The PPIX amount in thereaction solution to which (XII) metabolism solution (A1) was added wasmore than the PPIX amount in the reaction solution to which (XI) controlsolution (A1) was added.

(HPLC Analysis Condition 2)

-   column: SUMPAX ODS212 (Sumika Chemical Analysis Service)-   flow rate: 2 ml/minute-   detection wave length: fluorescent Ex:410 nm Em:630 nm-   eluent: 95:5 mixture of methanol and 1M ammonium acetate (pH5.7).    (3) Metabolism of Compound (XIII) by the Present Invention Protein    (A1)

Other than utilizing 5 ppm of compound (XIII) instead of 5 ppm ofcompound (XII), reaction solutions were prepared and maintainedsimilarly to the method described in Example 41(2). Similarly to Example41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residues were dissolved in100 μl of dimethylsulfoxide. The obtained solutions (hereinafter, thesolution derived from the reaction solution containing component A,component B, component C and 20 μl of supernatant fraction recovered inExample 4(2) is referred to as “(XIII) metabolism solution (A1)”;further, the solution derived from the reaction solution containing nocomponent A, no component B, no component C and no supernatant fractionrecovered in Example 4(2) is referred to as “(XIII) control solution(A1)”) were analyzed on a HPLC under the above analysis condition 1.Compared to the concentration of compound (XIII) detected from (XIII)control solution (A1), the concentration of compound (XIII) detectedfrom (XIII) metabolism solution (A1) was lower. Further a peak, whichwas not detected from the (XIII) control solution (A1), was detectedfrom the (XIII) metabolism solution (A1). Mass spectrometry wasconducted for the compound contained in such a peak. The mass of thecompound contained in such a peak was 14 smaller than the mass ofcompound (XIII).

Twenty microliters (20 μl) of a 128-fold dilution of the above (XIII)metabolism solution (A1) and 60 μl of the plastid fraction were mixed.In darkened conditions, 20 μl of PPO substrate solution were added andmaintained at 30° C. for 1.5 hours. Further, instead of said 20 μl ofthe 128-fold dilution of (XIII) metabolism solution (A1), a reactionsolution to which 20 μl of the 128-fold dilution of (XIII) controlsolution (A1) was added was prepared, and the PPO substrate solution wasadded and maintained similarly. Similar to Example 41(2), each of thereaction solutions after the maintenance were prepared and subjected toreverse phase HPLC analysis under the above analysis condition 2 tomeasure the amount of PPIX. The PPIX amount in the reaction solution towhich (XIII) metabolism solution (A1) was added was more than the PPIXamount in the reaction solution to which (XIII) control solution (A1)was added.

(4) Metabolism of Compound (XVI) by the Present Invention Protein (A1)

Other than utilizing 12.5 ppm of compound (XVI) instead of 5 ppm ofcompound (XII), reaction solutions were prepared and maintainedsimilarly to the method described in Example 41(2). Similarly to Example41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residues were dissolved in200 μl of 50 mM potassium phosphate buffer (pH7.0). The obtainedsolutions (hereinafter, the solution derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 4(2) is referred to as “(XVI)metabolism solution (A1)”; further, the solution derived from thereaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 4(2) isreferred to as “(XVI) control solution (A1)”) were analyzed on a HPLCunder the above analysis condition 1. Compared to the concentration ofcompound (XVI) detected from (XVI) control solution (A1), theconcentration of compound (XVI) detected from (XVI) metabolism solution(A1) was lower. Further a peak, which was not detected from the (XVI)control solution (A1), was detected from the (XVI) metabolism solution(A1).

Twenty microliters (20 μl) of a 8-fold dilution of the above (XVI)metabolism solution (A1) and 60 μl of the plastid fraction were mixed.In darkened conditions, 20 μl of PPO substrate solution were added andmaintained at 30° C. for 15 hours. Further, instead of said 20 μl of the8-fold dilution of (XVI) metabolism solution (A1), a reaction solutionto which 20 μl of the 8-fold dilution of (XVI) control solution (A1) wasadded was prepared, and the PPO substrate solution was added andmaintained similarly. Similar to Example 41(2), each of the reactionsolutions after the maintenance were prepared and subjected to reversephase HPLC analysis under the above analysis condition 2 to measure theamount of PPIX. The PPIX amount in the reaction solution to which (XVI)metabolism solution (A1) was added was more than the PPIX amount in thereaction solution to which (XVI) control solution (A1) was added.

(5) Metabolism of Compound (XVII) by the Present Invention Protein (A1)

Other than utilizing 12.5 ppm of compound (XVII) instead of 5 ppm ofcompound (XII), reaction solutions were prepared and maintainedsimilarly to the method described in Example 41(2). Similarly to Example41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residues were dissolved in200 μl of 50 mM potassium phosphate buffer (pH7.0). The obtainedsolutions (hereinafter, the solution derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 4(2) is referred to as “(XVII)metabolism solution (A1)”; further, the solution derived from thereaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 4(2) isreferred to as “(XVII) control solution (A1)”) were analyzed on a HPLCunder the above analysis condition 1. Compared to the concentration ofcompound (XVII) detected from (XVII) control solution (A1), theconcentration of compound (XVII) detected from (XVII) metabolismsolution (A1) was lower. Further a peak, which was not detected from the(XVII) control solution (A1), was detected from the (XVII) metabolismsolution (A1).

Twenty microliters (20 μl) of a 32-fold dilution of the above (XVII)metabolism solution (A1) and 60 μl of the plastid fraction were mixed.In darkened conditions, 2041 of PPO substrate solution were added andmaintained at 30° C. for 1.5 hours. Further, instead of said 20 μl ofthe 32-fold dilution of (XVII) metabolism solution (A1), a reactionsolution to which 20 μl of the 32-fold dilution of (XVII) controlsolution (A1) was added was prepared, and the PPO substrate solution wasadded and maintained similarly. Similar to Example 41(2), each of thereaction solutions after the maintenance were prepared and subjected toreverse phase HPLC analysis under the above analysis condition 2 tomeasure the amount of PPIX. The PPIX amount in the reaction solution towhich (XVII) metabolism solution (A1) was added was more than the PPIXamount in the reaction solution to which (XVII) control solution (A1)was added.

(6) Metabolism of Compound (VI) by the Present Invention Protein (A1)

E. coli JM109/pKSN657F was cultured overnight at 37° C. in 3 ml of TBmedium containing 50 μg/ml of ampicillin. A milliliter (1 ml) of theobtained culture medium was transferred to 100 ml of TB mediumcontaining 50 μg/ml of ampicillin and cultured at 26° C. When OD660reached about 0.5, 5-aminolevulinic acid was added to the finalconcentration of 500 μM, and the culturing was continued. Thirty (30)minutes thereafter, IPTG was added to a final concentration of 1 mM, andthere was further culturing for 20 hours.

The cells were recovered from the culture medium, washed with 0.1Mtris-HCl buffer (pH7.5) and suspended in 10 ml of 0.1M Tris-HCl buffercontaining 1% glucose. Compound (VI) was added to the obtained cellsuspension to a final concentration of 100 ppm and that was incubatedwith shaking at 30° C. At each of 0 hours after and 1 day after thestart of shaking, 2 ml of the cell suspension were fractioned. Fiftymicroliters (50 μl) of 2N HCl were added to each and those wereextracted with 2 ml of ethyl acetate. The obtained ethyl acetate layerswere analyzed on a HPLC under reaction condition 1. Compared to theconcentration of compound (VI) detected from the ethyl acetate layerprepared from the cell suspension at 0 hours after the start of shaking,the concentration of compound (VI) detected from the ethyl acetate laterprepared from the cell suspension at 1 day after the start of shakingwas lower. Further a peak, which was not detected from the ethyl acetatelayer prepared from the cell suspension at 0 hours after the start ofshaking, was detected from the ethyl acetate layer prepared from thecell suspension at 1 day after the start of shaking. Mass spectrometryof the compound contained in said peak was conducted. The mass of thecompound contained in said peak was 14 less than the mass of compound(VI).

(7) Metabolism of Compound (VIII) by the Present Protein (A1)

Other than utilizing compound (VIII) instead of compound (VI), there wasconducted in accordance with the method described in Example 41(6), aculturing of E. coli JM109/pKSN657F, preparation of the cell suspensionsolution, incubation with shaking of the cell suspension solution towhich compound (VIII) was added, reagent preparation from the cellsuspension solution and HPLC analysis of the reagents. Compared to theconcentration of compound (VIII) detected from the ethyl acetate layerprepared from the cell suspension at 0 hours after the start of shaking,the concentration of compound (VIII) detected from the ethyl acetatelayer prepared from the cell suspension at 1 day after the start ofshaking was lower Further two peaks, which were not detected from theethyl acetate layer prepared from the cell suspension at 0 hours afterthe start of shaking, were detected from the ethyl acetate layerprepared from the cell suspension at 1 day after the st of shaking. Massspectrometry of the compounds contained in said peaks were conducted.The mass of the compound contained in one of said peaks was 14 less andthe mass of the compound contained in the other peak was 28 less thanthe mass of compound (VIII).

(8) Metabolism of Compound (X) by the Present Invention Protein (A1)

Other than utilizing compound (X) instead of compound (VI), there wasconducted in accordance with the method described in Example 41(6), aculturing of E. coli JM109/pKSN657F, preparation of the cell suspensionsolution, shake culturing of the cell suspension solution to whichcompound (X) was added, reagent preparation from the cell suspensionsolution and HPLC analysis of the reagents. Compared to theconcentration of compound (X) detected from the ethyl acetate layerprepared from the cell suspension at 0 hours after the start of shaking,the concentration of compound (X) detected from the ethyl acetate laterprepared from the cell suspension at 1 day after the start of shakingwas lower. Further two peaks, which were not detected from the ethylacetate layer prepared from the cell suspension at 0 hours after thestart of shaking, were detected from the ethyl acetate layer preparedfrom the cell suspension at 1 day after the start of shaking. Massspectrometry of the compounds contained in said peaks was conducted. Themass of the compound contained in one of said peaks was 40 less and themass of the compound contained in the other peak was 54 less than themass of compound (X).

(9) Metabolism of Compound (XI) by the Present Invention Protein (A1)

Other than utilizing compound (XI) instead of compound (VI), there wasconducted in accordance with the method described in Example 41(6), aculturing of E. coli JM109/pKSN657F, preparation of the cell suspensionsolution, shake culturing of the cell suspension solution to whichcompound (XI) was added, reagent preparation from the cell suspensionsolution and HPLC analysis of the reagents. Compared to theconcentration of compound (XI) detected from the ethyl acetate layerprepared from the cell suspension at 0 hours after the start of shaking,the concentration of compound (XI) detected from the ethyl acetate layerprepared from the cell suspension at 1 day after the start of shakingwas lower. Further two peaks, which were not detected from the ethylacetate layer prepared from the cell suspension at 0 hours after thestart of shaking, were detected from the ethyl acetate layer preparedfrom the cell suspension at 1 day after the start of shaking. Massspectrometry of the compounds contained in said peaks was conducted. Themass of the compound contained in one of said peaks was 14 less and themass of the compound contained in the other peak was 16 less than themass of compound (XI).

Example 42 Metabolism of Compounds by the Present Invention Protein(A11)

(1) Metabolism of Compound (X) by the Present Invention Compound (A11)

Each of E. coli JM109/pKSN849AF and E. coli JM109/pKSN2 was culturedovernight at 37° C. in 3 ml of TB culture containing 50 μg/ml ofampicillin. A milliliter (1 ml) of the obtained culture mediums wastransferred to 100 ml of TB medium containing 50 μg/ml of ampicillin andcultured at 26° C., When OD660 reached about 0.5, 5-aminolevulinic acidwas added to the final concentration of 500 μM, and the culturing wascontinued. Thirty (30) minutes thereafter, IPTG was added to a finalconcentration of 1 mM, and there was further culturing for 18 hours.

The cells were recovered from the culture medium, washed with 0.1Mtris-HCl buffer (pH7.5) and suspended in 10 ml of 0.1M Tris-HCl buffercontaining 1% glucose. Compound (X) was added to the obtained cellsuspension to a final concentration of 25 ppm and that was incubatedwith shaking at 30° C. At each of 0 hours after and 4 days after thestart of shaking, 2 ml of the cell suspension were fractioned. Fiftymicroliters (50 μl) of 2N HCl were added to each and those wereextracted with 2 ml of ethyl acetate. The obtained ethyl acetate layerswere analyzed on a HPLC under reaction condition 1. Compared to theconcentration of compound (X) detected from the ethyl acetate layerprepared from the JM109/pKSN2 cell suspension, the concentration ofcompound (X) detected from the ethyl acetate layer prepared from theJM109/pKSN849AF cell suspension was lower. Further 3 peaks, which werenot detected from the ethyl acetate layer prepared from the JM109/pKSN2cell suspension, were detected from the ethyl acetate layer preparedfrom the JM109/pKSN849AF cell suspension. Of the 3 peaks, the elutiontime in the HPLC of 1 of the peaks matched with the elution time of apeak of a compound that has a mass of 40 less than compound (X) detectedin Example 41(8). Further, the elution time in the HPLC of another peakmatched with the elution time of a peak of a compound that has a mass of54 less than compound (X) detected in Example 41(8).

After drying, respectively, 1 ml of the ethyl acetate layer preparedfrom the above JM109/pKSN2 cell suspension and 1 ml of the ethyl acetatelayer prepared from the above JM109/pKSN849AF cell suspension, theresidues were dissolved in 1 ml of dimethylsulfoxide (hereinafter, thesolution derived from the ethyl acetate layer prepared fromJM109/pKSN849AF is referred to as “(X) metabolism solution (A11)”;further, the solution derived from the ethyl acetate layer prepared fromJM109/pKSN2 cell suspension is referred to as “(X) control solution(A11)”).

Twenty microliters (20 μl) of a 128-fold dilution of the above (Xmetabolism solution (A11) and 60 μl of the plastid fraction were mixed.In darkened conditions, 20 μl of PPO substrate solution were added andmaintained at 30° C. for 1.5 hours. Further, instead of said 20 μl ofthe 128-fold dilution of (X) metabolism solution (A11), a reactionsolution to which 20 μl of the 128-fold dilution of (X) control solution(A11) was added was prepared, and the PPO substrate solution was addedand maintained similarly. Similar to Example 41(2), each of the reactionsolutions after the maintenance were prepared and subjected to reversephase HPLC analysis under the above analysis condition 2 to measure theamount of PPIX. The PPIX amount in the reaction solution to which (X)metabolism solution (A11) was added was more than the PPIX amount in thereaction solution to which (X) control solution (A11) was added.

(2) Metabolism of Compound (XII) by the Present Invention Protein (A11)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 32(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solutions were prepared and maintained inaccordance with the method described in Example 41(2). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in100 μl of 50 mM potassium phosphate buffer (pH7.0). The obtainedsolutions (hereinafter, the solution derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 32(2) is referred to as “(XII)metabolism solution (A11)”; further, the solution derived from thereaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 32(2) isreferred to as “(XII) control solution (A11)”) were analyzed on a HPLCunder the above analysis condition 1. Compared to the concentration ofcompound (XII) detected from (XII) control solution (A11), theconcentration of compound (XII) detected from (XII) metabolism solution(A11) was lower. Further a peak, which was not detected from the (XII)control solution (A11), was detected from the (XII) metabolism solution(A11). The elution time of said peak on the HPLC matched an elution timeof a peak of a compound in which the mass is 14 less than said compound(XII) detected from (XI) metabolism solution (A1) in Example 41(2).

(3) Metabolism of Compound (XII) by the Present Invention Protein (A11)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 32(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solutions were prepared and maintained inaccordance with the method described in Example 41(3). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in100 μl of dimethylsulfoxide. The obtained solutions (hereinafter, thesolution derived from the reaction solution containing component A,component B, component C and 20 μl of supernatant fraction recovered inExample 32(2) is referred to as “(XIII) metabolism solution (A11)”;further, the solution derived from the reaction solution containing nocomponent A, no component B, no component C and no supernatant fractionrecovered in Example 32(2) is referred to as “(XIII) control solution(A11)”) were analyzed on a HPLC under the above analysis condition 1.Compared to the concentration of compound (XIII) detected from (XII)control solution (A11), the concentration of compound (XIII) detectedfrom (XIII) metabolism solution (A11) was lower. Further a peak, whichwas not detected from the (XIII) control solution (A11), was detectedfrom the (XIII) metabolism solution (A11). The elution time of said peakon the HPLC matched an elution time of a peak of a compound in which themass is 14 less than said compound (XIII) detected from (XIII)metabolism solution (A11) in Example 41(3).

(4) Metabolism of Compound (XVI) by the Present Invention Protein (A11)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 32(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solutions were prepared and maintained inaccordance with the method described in Example 41(4). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in200 μl of 50 mM potassium phosphate buffer (pH7.0). The obtainedsolutions (hereinafter, the solution derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 32(2) is referred to as “(XVI)metabolism solution (A11)”; further, the solution derived from thereaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 32(2) isreferred to as “(XVI) control solution (A11)”) were analyzed on a HPLCunder the above analysis condition 1. Compared to the concentration ofcompound (XVI) detected from (XVI) control solution (A11), theconcentration of compound (XVI) detected from (XVI) metabolism solution(A11) was lower. Further a peak, which was not detected from the (XVI)control solution (A11), was detected from the (XVI) metabolism solution(A11). The elution time of said peak on the HPLC matched an elution timeof a peak in Example 41(4) which was detected from (XVI) metabolismsolution (A11) and not detected in (XVI) control solution (A11).

(5) Metabolism of Compound (XVII) by the Present Invention Protein (A11)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 32(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solutions were prepared and maintained inaccordance with the method described in Example 41(5). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in200 μl of 50 mM potassium phosphate buffer (pH7.0). The obtainedsolutions (hereinafter, the solution derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 32(2) is referred to as“(XVII) metabolism solution (A11)”; further, the solution derived fromthe reaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 32(2) isreferred to as “(XVII) control solution (A11)”) were analyzed on a HPLCunder the above analysis condition 1. Compared to the concentration ofcompound (XVII) detected from (XVII) control solution (A11), theconcentration of compound (XVII) detected from (XVII) metabolismsolution (A11) was lower. Further a peak, which was not detected fromthe (XVII) control solution (A11), was detected from the (XVII)metabolism solution (A11). The elution time of said peak on the HPLCmatched an elution time of a peak in Example 41(5) which was detectedfrom (XVII) metabolism solution (A1) and riot detected in (XVII) controlsolution (A1).

Example 43 Metabolism of Compounds by the Present Invention Protein(A2), (A3), (A12), (A13), (A14) or (A15) or the Present Protein (A10)

(1) Metabolism of Compound (XII) by the Present Invention Protein (A2)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 7(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solutions were prepared and maintained inaccordance with the method described in Example 41(2). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in100 μl of 50 mM potassium phosphate buffer (pH7.0). The obtainedsolutions (hereinafter, the solution derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 7(2) is referred to as “(XII)metabolism solution (A2)”; further, the solution derived from thereaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 7(2) isreferred to as “(XII) control solution (A2)”) were analyzed on a HPLCunder the above analysis condition 1. Compared to the concentration ofcompound (XII) detected from (XII) control solution (A2), theconcentration of compound (XII) detected from (XII) metabolism solution(A2) was lower. Further a peak, which was not detected from the (XII)control solution (A2), was detected from the (XII) metabolism solution(A2). The elution time of said peak on the HPLC matched an elution timeof a peak of a compound in which the mass is 14 less than said compound(XII) detected from (XII) metabolism solution (A1) in Example 41(2).

(2) Metabolism of Compound (XII) by the Present Invention Protein (A3)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 12(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solutions were prepared and maintained inaccordance with the method described in Example 41(2). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in100 μl of 50 mM potassium phosphate buffer (pH7.0). The obtainedsolutions (hereinafter, the solution derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 12(2) is referred to as “(XII)metabolism solution (A3)”; further, the solution derived from thereaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 7(2) isreferred to as “(XII) control solution (A3)”) were analyzed on a HPLCunder the above analysis condition 1. Compared to the concentration ofcompound (XII) detected from (XII) control solution (A3), theconcentration of compound (XII) detected from (XII) metabolism solution(A3) was lower. Further a peak, which was not detected from the (XII)control solution (A3), was detected from the (XII) metabolism solution(A3). The elution time of said peak on the HPLC matched an elution timeof a peak of a compound in which the mass is 14 less than said compound(XII) detected from (XII) metabolism solution (A1) in Example 41(2).

(3) Metabolism of Compound (XII) by the Present Protein (A10)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 10(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solutions were prepared and maintained inaccordance with the method described in Example 41(2). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in100 μl of 50 mM potassium phosphate buffer (pH7.0). The obtainedsolutions (hereinafter, the solution derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 10(2) is referred to as “(XII)metabolism solution (A10)”; further, the solution derived from thereaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 12(3) isreferred to as “(XII) control solution (A10)”) were analyzed on a HPLCunder the above analysis condition 1. Compared to the concentration ofcompound (XII) detected from (XII) control solution (A10), theconcentration of compound (XII) detected from (XII) metabolism solution(A10) was lower. Further a peak, which was not detected from the (XII)control solution (A10), was detected from the (XII) metabolism solution(A10). The elution time of said peak on the HPLC matched an elution timeof a peak of a compound in which the mass is 14 less than said compound(XII) detected from (XII) metabolism solution (A1) in Example 41(2).

(4) Metabolism of Compound (XII) by the Present Invention Protein (A12)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 34(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solutions were prepared and maintained inaccordance with the method described in Example 41(2). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in100 μl of 50 mM potassium phosphate buffer (pH7.0). The obtainedsolutions (hereinafter, the solution derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 34(2) is referred to as “(XII)metabolism solution (A12)”; further, the solution derived from thereaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 34(2) isreferred to as “(XII) control solution (A12)”) were analyzed on a HPLCunder the above analysis condition 1. Compared to the concentration ofcompound (XII) detected from (XII) control solution (A12), theconcentration of compound (XII) detected from (XII) metabolism solution(A12) was lower. Further a peak, which was not detected from the (XII)control solution (A12), was detected from the (XII) metabolism solution(A12). The elution time of said peak on the HPLC matched an elution timeof a peak of a compound in which the mass is 14 less than said compound(XII) detected from (XII) metabolism solution (A1) in Example 41(2).

(5) Metabolism of Compound (XII) by the Present Invention Protein (A13)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 36(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solutions were prepared and maintained inaccordance with the method described in Example 41(2). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in100 μl of 50 mM potassium phosphate buffer (pH7.0). The obtainedsolutions (hereinafter, the solution derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 36(2) is referred to as “(XII)metabolism solution (A13)”; further, the solution derived from thereaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 36(2) isreferred to as “(XII) control solution (A13)”) were analyzed on a HPLCunder the above analysis condition 1. Compared to the concentration ofcompound (XII) detected from (XII) control solution (A13), theconcentration of compound (XII) detected from (XII) metabolism solution(A13) was lower. Further a peak, which was not detected from the (XII)control solution (A13), was detected from the (XII) metabolism solution(A13). The elution time of the said peak on the HPLC matched an elutiontime of a peak of a compound in which The mass is 14 less than saidcompound (XII) detected from (XII) metabolism solution (A1) in Example41(2).

(6) Metabolism of Compound (XII) by the Present Invention Protein (A14)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 38(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solutions were prepared and maintained inaccordance with the method described in Example 41(2). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in100 μl of 50 mM potassium phosphate buffer (pH7.0). The obtainedsolutions (hereinafter, the solution derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 38(2) is referred to as “(XII)metabolism solution (A14)”; further, the solution derived from thereaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 38(2) isreferred to as “(XII) control solution (A14)”) were analyzed on a HPLCunder the above analysis condition 1. Compared to the concentration ofcompound (XII) detected from (XII) control solution (A14), theconcentration of compound (XII) detected from (XII) metabolism solution(A14) was lower. Further a peak, which was not detected from the (XII)control solution (A14), was detected from the (XII) metabolism solution(A14). The elution time of said peak on the HPLC matched an elution timeof a peak of a compound in which the mass is 14 less than said compound(XII) detected from (XII) metabolism solution (A1) in Example 41(2).

(7) Metabolism of Compound (XII) by the Present Invention Protein (A15)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 40(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solutions were prepared and maintained inaccordance with the method described in Example 41(2). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in100 μl of 50 mM potassium phosphate buffer (pH7.0). The obtainedsolutions (hereinafter, the solution derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 40(2) is referred to as “(XII)metabolism solution (A15)”; further, the solution derived from thereaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 40(2) isreferred to as “(XII) control solution (A15)”) were analyzed on a HPLCunder the above analysis condition 1. Compared to the concentration ofcompound (XII) detected from (XI) control solution (A15), theconcentration of compound (XII) detected from (XII) metabolism solution(A15) was lower. Further a peak, which was not detected from the (XII)control solution (A15), was detected from the (XII) metabolism solution(A15). The elution time of said peak on the HPLC matched an elution timeof a peak of a compound in which the mass is 14 less than said compound(XII) detected from (XII) metabolism solution (A1) in Example 41(2)

(8) Metabolism of Compound (XIII) by the Present Invention Protein (A2)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 7(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solutions were prepared and maintained inaccordance with the method described in Example 41(3). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in100 μl of dimethylsulfoxide. The obtained solutions (hereinafter, thesolution derived from the reaction solution containing component A,component B, component C and 20 μl of supernatant fraction recovered inExample 7(2) is referred to as “(XIII) metabolism solution (A2)”;farther, the solution derived from the reaction solution containing nocomponent A, no component B, no component C and no supernatant fractionrecovered in Example 7(2) is referred to as “(XIII) control solution(A2)”) were analyzed on a HPLC under the above analysis condition 1.Compared to the concentration of compound (XIII) detected from (XIII)control solution (A2), the concentration of compound (XIII) detectedfrom (XIII) metabolism solution (A2) was lower. Further a peak, whichwas not detected from the (XIII) control solution (A2), was detectedfrom the (XIII) metabolism solution (A2). The elution time of said peakon the HPLC matched an elution time of a peak of a compound in which themass is 14 less than said compound (XIII) detected from (XIII)metabolism solution (A1) in Example 41(3).

(9) Metabolism of Compound (XI) by the Present Invention Protein (A3)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 12(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solutions were prepared and maintained inaccordance with the method described in Example 41(3). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in100 μl of dimethylsulfoxide. The obtained solutions (hereinafter, thesolution derived from the reaction solution containing component A,component B, component C and 20 μl of supernatant fraction recovered inExample 12(2) is referred to as “(XIII) metabolism solution (A3)”;further, the solution derived from the reaction solution containing nocomponent A, no component B, no component C and no supernatant fractionrecovered in Example 12(2) is referred to as “(XIII) control solution(A3)”) were analyzed on a HPLC under the above analysis condition 1.Compared to the concentration of compound (XIII) detected from (XIII)control solution (A3), the concentration of compound (III) detected from(XIII) metabolism solution (A3) was lower. Further a peak, which was notdetected from the (XIII) control solution (A3), was detected from the(XIII) metabolism solution (A3). The elution time of said peak on theHPLC matched an elution time of a peak of a compound in which the massis 14 less than said compound (XIII) detected from (XIII) metabolismsolution (A1) in Example 41(3).

(10) Metabolism of Compound (XIII) by the Present Protein (A10)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 10(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solutions were prepared and maintained inaccordance with the method described in Example 41(3). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in100 μl of dimethylsulfoxide. The obtained solutions (hereinafter, thesolution derived from the reaction solution containing component A,component B, component C and 20 μl of supernatant fraction recovered inExample 10(2) is referred to as “(XIII) metabolism solution (A10)”;further, the solution derived from the reaction solution containing nocomponent A, no component B, no component C and no supernatant fractionrecovered in Example 10(2) is referred to as “(XIII) control solution(A10)”) were analyzed on a HPLC under the above analysis condition 1.Compared to the concentration of compound (XII) detected from (XIII)control solution (A10), the concentration of compound (XIII) detectedfrom (XIII) metabolism solution (A10) was lower. Further a peak, whichwas not detected from the (XIII) control solution (A10), was detectedfrom the (XIII) metabolism solution (A10). The elution time of the saidpeak on the HPLC matched an elution time of a peak of a compound inwhich the mass is 14 less than said compound (XIII) detected from (XIII)metabolism solution (A1) in Example 41(3).

(11) Metabolism of Compound (XII) by the Present Invention Protein (A12)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 34(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solutions were prepared and maintained inaccordance with the method described in Example 41(3). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in100 μl of dimethylsulfoxide. The obtained solutions (hereinafter, thesolution derived from the reaction solution containing component A,component B, component C and 20 μl of supernatant fraction recovered inExample 34(2) is referred to as “(XIII) metabolism solution (A12)”;further, the solution derived from the reaction solution containing nocomponent A, no component B, no component C and no supernatant fractionrecovered in Example 34(2) is referred to as “(XIII) control solution(A12)”) were analyzed on a HPLC under the above analysis condition 1.Compared to the concentration of compound (XIII) detected from (XIII)control solution (A12), the concentration of compound (XIII) detectedfrom (XIII) metabolism solution (A12) was lower. Further a peak, whichwas not detected from the (XIII) control solution (A12), was detectedfrom the (XIII) metabolism solution (A12). The elution time of said peakon the HPLC matched an elution time of a peak of a compound in which themass is 14 less than said compound (XIII) detected from (XIII)metabolism solution (A1) in Example 41(3).

(12) Metabolism of Compound (XIII) by the Present Invention Protein(A13)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 36(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solutions were prepared and maintained inaccordance with the method described in Example 41(3). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in100 μl of dimethylsulfoxide. The obtained solutions (hereinafter, thesolution derived from the reaction solution containing component A,component B, component C and 20 μl of supernatant fraction recovered inExample 36(2) is referred to as “(XIII) metabolism solution (A13)”;further, the solution derived from the reaction solution containing nocomponent A, no component B, no component C and no supernatant fractionrecovered in Example 36(2) is referred to as “(XIII) control solution(A13)”) were analyzed on a HPLC under the above analysis condition 1.Compared to the concentration of compound (XIII) detected from (XIII)control solution (A13), the concentration of compound (XIII) detectedfrom (XIII) metabolism solution (A13) was lower. Further a peak, whichwas not detected from the (XIII) control solution (A13), was detectedfrom the (XIII) metabolism solution (A13). The elution time of said peakon the HPLC matched an elution time of a peak of a compound in which themass is 14 less than said compound (XIII) detected from (XIII)metabolism solution (A1) in Example 41(3).

(13) Metabolism of Compound (XIII) by the Present Invention Protein(A14)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 38(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solution were prepared and maintained inaccordance with the method described in Example 41(3). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in100 μl of dimethylsulfoxide. The obtained solutions (hereinafter, thesolution derived from the reaction solution containing component A,component B, component C and 20 μl of supernatant fraction recovered inExample 38(2) is referred to as “(XII) metabolism solution (A14)”;further, the solution derived from the reaction solution containing nocomponent A, no component B, no component C and no supernatant fractionrecovered in Example 38(2) is referred to as “(XIII) control solution(A14)”) were analyzed on a HPLC under the above analysis condition 1.Compared to the concentration of compound (XIII) detected from (XIII)control solution (A14), the concentration of compound (XIII) detectedfrom (XIII) metabolism solution (A14) was lower. Further a peak, whichwas not detected from the (XIII) control solution (A14), was detectedfrom the (XIII) metabolism solution (A14). The elation time of said peakon the HPLC matched an elution time of a peak of a compound in which themass is 14 less than said compound (XIII) detected from (XIII)metabolism solution (A1) in Example 41(3).

(14) Metabolism of Compound (XIII) by the Present Invention Protein(A15)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 40(2) instead of 20 μl of the supernatant fraction recovered inExample 4(2), the reaction solutions were prepared and maintained inaccordance with the method described in Example 41(3). Similar toExample 41(2), each of the reaction solutions after the maintenance wasextracted with ethyl acetate and the obtained residue was dissolved in100 μl of dimethylsulfoxide. The obtained solutions (hereinafter, thesolution derived from the reaction solution containing component A,component B, component C and 20 μl of supernatant fraction recovered inExample 40(2) is referred to as “(XIII) metabolism solution (A15)”;further, the solution derived from the reaction solution containing nocomponent A, no component B, no component C and no supernatant fractionrecovered in Example 40(2) is referred to as “(XIII) control solution(A15)”) were analyzed on a HPLC under the above analysis condition 1.Compared to the concentration of compound (XIII) detected from (XIII)control solution (A15), the concentration of compound (XIII) detectedfrom (XIII) metabolism solution (A15) was lower. Further a peak, whichwas not detected from the (XII) control solution (A15), was detectedfrom the (XIII) metabolism solution (A15). The elution time of said peakon the HPLC matched an elution time of a peak of a compound in which themass is 14 less than said compound (XIII) detected from (XIII)metabolism solution (A1) in Example 41(3).

Example 44 Preparation of the Present Invention Antibody (A) Recognizingthe Present Invention Protein (A1) (Hereinafter Referred to as “PresentInvention Antibody (A1)”)

(1) Preparation of the Extract of an E. coli Expressing the PresentInvention Protein (A1)

In accordance with the method described in Example 4(2), E. coliJM109/pKSN657F, which expresses the present invention protein (A1), waspre-cultured overnight and then cultured in 1 L of TB medium containing50 μg/ml of ampicillin. After recovering and disrupting the cells,supernatant fractions (E. coli pKSN657F extract) were prepared from theobtained cell lysate solution.

(2) Purification of the Present Invention Protein (A1)

The present invention protein (A1) was purified according to the methoddescribed in Example 2(4) by subjecting the supernatant fractionobtained in Example 44(1) (E. coli pKSN657F extract) in turn to a HiloadHiLoad26/10 Q Sepharose IP column and then a Bio-Scale CeramicHydroxyapatite, Type I column CHT10-1 column. The purified fractionswere analyzed on a 10% to 20% SDS-PAGE, to confirm that those werefractions of only the present invention protein (A1).

(3) Preparation of the Present Invention Antibody (A1)

The present invention protein (A1) prepared in Example 44(2) wasdissolved in 0.05M potassium phosphate buffer (pH7.0) so that theconcentration was 1 mg/ml. Forty microliters (40 μl) of RAS (MPL(Monophosphoryl lipid A)+TDM (Synthetic Trehalose Dicorynomycolate)+CWS(Cell Wall Skeleton) Adjuvant System (Sigma Company)) already incubatedat 42° C. to 43° C. was added and well mixed into 2 ml of the obtainedsolution. The obtained mixture was administered, respectively, to NewZealand White rabbits (female, 14 weeks old, average of 2.4 kg) at 1 mlper rabbit. As such, 100 μl was injected subcutaneously at 10 locationson the back. About ½ of the amount of the first administration wasadministered after each of 3 weeks and 5 weeks. During such time, theantibody titer was measured by sampling the blood from a ear vein of therabbit. Since the antibody titer increased after the thirdadministration, the immunized rabbit at 2 weeks after the thirdadministration was exsanguinated from the neck. The obtained blood wasadded into a Separapit Tube (Sekisui Chemical Company), incubated at 37°C. for 2 hours and was then centrifuged (3000 rpm, 20 minutes, roomtemperature). The antiserum (containing the present invention antibody(A1)) was obtained by recovering the supernatant.

Example 45 Detection of the Present Protein by the Present InventionAntibody (A1) and Detection of a Cell Expressing the Present Protein

An immunoblot was conducted by utilizing the present invention antibody(A1) obtained in Example 44 with each of the E. coli extracts. There wasa SDS polyacrylamide electrophoresis (40 mA, 1 hour) of the E. colipKSN657F extract obtained in Example 4(2) (containing about 0.5 pmol ofthe present invention protein (A1), containing about 0.78 mg ofprotein); the E. coli pKSN2 extract obtained in Example 4(2) (containingabout 0.78 mg of protein) the E. coli pKSN923F extract obtained inExample 7(2) (containing about 2 pmol of the present invention protein(A2)); the E. coli pKSN671F extract obtained in Example 12(2)(containing about 2 pmol of the present invention protein (A3)); the E.coli pKSN646F extract obtained in Example 27(2) (containing about 2 pmolof the present invention protein (A4)); the E. coli pKSN11796F extractobtained in Example 10(2) (containing about 2 pmol of the presentprotein (A10)); the E. coli pKSNSCA extract obtained in Example 14(2)(containing about 2 pmol of the present protein (A9)); the E. colipKSN849AF extract obtained in Example 32(2) (containing about 2 pmol ofthe present invention protein (A11)); the E. coli pKSN1618F extractobtained in Example 34(2) (containing about 2 pmol of the presentinvention protein (A12)); the E. coli pKSN474F extract obtained inExample 36(2) (containing about 2 pmol of the present invention protein(A13)); the E. coli pKSN1491AF extract obtained in Example 38(2)(containing about 2 pmol of the present invention protein (A14)); andthe E. coli pKSN1555AF extract obtained in Example 40(2) (containingabout 2 pmol of the present invention protein (A15)). A PVDF membranewas placed on the gel. The proteins in the gel were transferred onto thePVDF membrane by a treatment with a BioRad blotting device at 4° C., 30Vfor 2 hours, while in the condition of being soaked in transfer buffer(25 mM Tris, 192 mM glycine, 10% methanol). After washing with TBS+Tween20 solution (50 mM Tris-HCl (pH7.5), 200 mM NaCl, 0.05% Tween 20), theobtained PVDF membrane was incubated for 30 minutes in TBS+Tween 20solution containing 3% BSA and was then utilized for a reaction with theabove antiserum diluted 30,000 fold for 30 minutes in TBS+Tween 20solution containing 3% BSA. After the reaction, the PVDF membrane waswashed twice with TBS+Tween 20 solution. The PVDF membrane was thenutilized for a reaction in TBS+Tween 20 solution containing 3% BSA for30 minutes with a 3000 fold dilution of anti-rabbit IgG goat anti-serumlabeled with alkaline phosphatase (Santa Cruz Biotechnology Company).After the reaction, the PVDF membrane was washed twice with TBS+Tween 20solution and was soaked in NBT-BCIP solution (Sigma Company). There wasdetected a stain for a band corresponding to each of the presentinvention proteins (A1), (A2), (A3), (A4), (A11), (A12), (A13), (A14)and (A15) as well as the present proteins (A9) and (A10). No stainedband was detected with the reagent of E. coli pKSN2 extract (containingabout 0.78 mg of protein) obtained in Example 4(2).

Example 46 Preparation and Expression of the Present Invention DNA (A1)in which the Codon Usage has been Adjusted for Expression in Soybean(Hereinafter Referred to as the “Present Invention DNA (A1)S”)

(1) Preparation of the Present Invention DNA (A1)S

PCR was conducted with Pyrobest DNA polymerase (Takara Shuzo Company)according to the attached manual, by utilizing a primer having anucleotide sequence shown in SEQ ID NO: 192 and a primer having anucleotide sequence shown in SEQ ID NO: 213. An aliquot of the obtainedPCR product was utilized as a template for a PCR conducted similarlyutilizing a primer having the nucleotide sequence shown in SEQ ID NO:191 and a primer having the nucleotide sequence shown in SEQ ID NO: 212.Further, an aliquot of that PCR product was utilized as a template for aPCR conducted similarly utilizing a primer having the nucleotidesequence shown in SEQ ID NO: 190 and a primer having the nucleotidesequence shown in SEQ ID NO: 211. The obtained reaction solution wasdesignated as reaction solution 1.

PCR was conducted with Pyrobest DNA polymerase (Takara Shuzo Company)according to the attached manual, by utilizing a primer having anucleotide sequence shown in SEQ ID NO: 195 and a primer having anucleotide sequence shown in SEQ ID NO: 210. An aliquot of the obtainedPCR product was utilized as a template for a PCR conducted similarlyutilizing a primer having the nucleotide sequence shown in SEQ ID NO:194 and a primer having the nucleotide sequence shown in SEQ ID NO: 209.Further, an aliquot of that PCR product was utilized as a template for aPCR conducted similarly utilizing a primer having the nucleotidesequence shown in SEQ ID NO: 193 and a primer having the nucleotidesequence shown in SEQ ID NO: 208. The obtained reaction solution wasdesignated as reaction solution 2.

PCR was conducted with Pyrobest DNA polymerase (Takara Shuzo Company)according to the attached manual by utilizing a primer having anucleotide sequence shown in SEQ ID NO: 198 and a primer having anucleotide sequence shown in SEQ ID NO: 207. An aliquot of the obtainedPCR product was utilized as a template for a PCR conducted similarlyutilizing a primer having the nucleotide sequence shown in SEQ ID NO:197 and a primer having the nucleotide sequence shown in SEQ ID NO: 206.Further, an aliquot of that PCR product was utilized as a template for aPCR conducted similarly utilizing a primer having the nucleotidesequence shown in SEQ ID NO: 196 and a primer having the nucleotidesequence shown in SEQ ID NO: 205. The obtained reaction solution wasdesignated as reaction solution 3.

PCR was conducted with Pyrobest DNA polymerase (Takara Shuzo Company)according to the attached manual, by utilizing a primer having anucleotide sequence shown in SEQ ID NO: 201 and a primer having anucleotide sequence shown in SEQ ID NO: 204. An aliquot of the obtainedPCR product was utilized as a template for a PCR conducted similarlyutilizing a primer having the nucleotide sequence shown in SEQ ID NO:200 and a primer having the nucleotide sequence shown in SEQ ID NO: 203.Further, an aliquot of that PCR product was utilized as a template for aPCR conducted similarly utilizing a primer having the nucleotidesequence shown in SEQ ID NO: 199 and a primer having the nucleotidesequence shown in SEQ ID NO: 202. The obtained reaction solution wasdesignated as reaction solution 4.

The reaction solutions 1 to 4 obtained in such a way were mixed. PCR wasconducted with Pyrobest DNA polymerase (Takara Shuzo Company) accordingto the attached manual, by utilizing as a template an aliquot of themixture thereof and by utilizing a primer having a nucleotide sequenceshown in SEQ ID NO: 190 and a primer having a nucleotide sequence shownin SEQ ID NO: 202. The nucleotide sequence of the amplified DNA wasconfirmed. There was obtained a DNA having a sequence in which thenucleotide sequence 5′cat-3′ is connected upstream of the 5′ terminusand the nucleotide sequence 5′-aagctt-3′ is connected downstream of the3′ terminus of the nucleotide sequence shown in SEQ ID NO: 214.

The codon usage of the present invention DNA (A1) having the nucleotidesequence shown in SEQ ID NO: 6 (GC content of 70.58%) is shown in Table22 and Table 23. The codon usage of soybean (GC content of 46.12%, CodonUsage Database publised by Kazusa DNA Research Institute(http//:www.kazusa.orjp/codon)) is shown in Table 24 and Table 25. Thecodon usage of the present invention DNA (A1) having the nucleotidesequence shown in SEQ ID NO: 214 (GC content of 51.59%) is shown inTable 26 and Table 27. TABLE 22 codon % codon % TTT 0.00 TCT 0.00 TTC3.18 TCC 1.71 TTA 0.00 TCA 0.00 TTG 1.22 TCG 2.20 CTT 0.00 CCT 0.00 CTC3.67 CCC 4.16 CTA 0.00 CCA 0.00 CTG 7.09 CCG 2.69 ATT 0.24 ACT 0.24 ATC4.16 ACC 2.69 ATA 0.00 ACA 0.24 ATG 2.69 ACG 1.96 GTT 0.24 GCT 0.00 GTC3.67 GCC 7.58 GTA 0.00 GCA 0.49 GTG 3.18 GCG 3.42

TABLE 23 codon % codon % TAT 0.00 TGT 0.24 TAC 1.47 TGC 0.98 TAA 0.00TGA 0.00 TAG 0.24 TGG 0.98 CAT 0.24 CGT 1.22 CAC 2.20 CGC 4.40 CAA 0.24CGA 0.24 CAG 2.93 CGG 4.16 AAT 0.00 AGT 0.00 AAC 1.22 AGC 0.49 AAA 0.24AGA 0.00 AAG 0.98 AGG 0.00 GAT 0.98 GGT 0.98 GAC 7.82 GGC 3.42 GAA 0.73GGA 0.24 GAG 5.38 GGG 1.22

TABLE 24 codon % codon % TTT 2.03 TCT 1.71 TTC 2.09 TCC 1.21 TTA 0.82TCA 1.45 TTG 2.21 TCG 0.044 CTT 2.36 CCT 2.00 CTC 1.66 CCC 1.01 CTA 0.82CCA 2.05 CTG 1.22 CCG 0.40 ATT 2.61 ACT 1.78 ATC 1.64 ACC 1.49 ATA 1.27ACA 1.51 ATG 2.27 ACG 0.41 GTT 2.67 GCT 2.81 GTC 1.24 GCC 1.69 GTA 0.73GCA 2.27 GTG 2.20 GCG 0.59

TABLE 25 codon % codon % TAT 1.61 TGT 0.72 TAC 1.53 TGC 0.75 TAA 0.11TGA 0.09 TAG 0.06 TGG 1.21 CAT 1.33 CGT 0.72 CAC 1.09 CGC 0.63 CAA 2.04CGA 0.38 CAG 1.71 CGG 0.27 AAT 2.10 AGT 1.21 AAC 2.27 AGC 1.08 AAA 2.63AGA 1.42 AAG 3.83 AGG 1.35 GAT 3.29 GGT 2.17 GAC 2.06 GGC 1.38 GAA 3.35GGA 2.23 GAG 3.46 GGG 1.29

TABLE 26 codon % codon % TTT 1.71 TCT 0.98 TTC 1.47 TCC 0.73 TTA 0.98TCA 0.98 TTG 2.93 TCG 0.24 CTT 3.18 CCT 2.44 CTC 2.20 CCC 1.22 CTA 0.98CCA 2.69 CTG 1.71 CCG 0.49 ATT 2.20 ACT 1.71 ATC 1.22 ACC 1.47 ATA 0.98ACA 1.47 ATG 2.69 ACG 0.49 GTT 2.93 GCT 4.16 GTC 1.22 GCC 2.69 GTA 0.73GCA 3.67 GTG 2.20 GCG 0.98

TABLE 27 codon % codon % TAT 0.73 TGT 0.73 TAC 0.73 TGC 0.49 TAA 0.00TGA 0.00 TAG 0.24 TGG 0.98 CAT 1.47 CGT 1.47 CAC 0.98 CGC 1.47 CAA 1.71CGA 0.73 CAG 1.47 CGG 0.49 AAT 0.73 AGT 0.73 AAC 0.49 AGC 0.73 AAA 0.49AGA 2.93 AAG 0.73 AGG 2.93 GAT 5.38 GGT 1.71 GAC 3.42 GGC 1.22 GAA 2.69GGA 1.96 GAG 3.42 GGG 0.98(2) Production of a Transformed E. coli Having the Present InventionProtein (A1)S

The DNA having the nucleotide sequence shown in SEQ ID NO: 214 obtainedin Example 46(1) was digested with restriction enzymes NdeI and HindIII.The obtained DNA and the plasmid pKSN2 digested with NdeI and HindIIIwere ligated to obtain a plasmid in which the DNA having the nucleotidesequence shown in SEQ ID NO: 214 is inserted between the NdeI site andthe HindIII site of pKSN2 (hereinafter referred to as “pKSN657 soy”).Said plasmid was introduced into E. coli JM109. The obtained E. colitransformant was designated JM109/pKSN657soy.

(3) Expression of the Present Invention Protein (A1) in E. coli andRecovery of Said Protein

Similarly to Example 4(2), each of E. coli JM109/pKSN657soy obtained inExample 46(2) and E. coli JM109/pKSN657 obtained in Example 4(1) wascultured. The cells were recovered. Cell lysate solutions were prepared.Under the method described in Example 4(2), supernatant fractions wereprepared from the cell lysate solutions (hereinafter, the supernatantfraction obtained from E. coli JM109/pKSN657soy is referred to as “E.coli pKSN849soy extract” and the supernatant fraction obtained from E.coli JM109/pKSN657 is referred to as “E. coli pKSN657 extract”). Theamount of P450 per the protein amount contained in E. coli pKSN657soyextract was compared to and was higher than the amount of P450 per theprotein amount contained in E. coli pKSN657 extract.

Example 47 Introduction of the Present Invention DNA (A1)S into a Plant

(1) Construction of a Chloroplast Expression Plasmid Containing thePresent Invention DNA (A1)S for Direct Introduction—Part 1

A plasmid containing a chimeric DNA in which the present invention DNA(A1)S was connected immediately after the nucleotide sequence encodingthe chloroplast transit peptide of soybean (cv. Jack) RuBPC smallsubunit without a change of frames in the codons was constructed as aplasmid for introducing the present invention DNA (A1)S into a plantwith the particle gun method.

First, DNA comprising the nucleotide sequence shown in SEQ ID NO: 214was amplified by PCR. The PCR was conducted by utilizing as a templatepKSN657soy obtained in Example 46(2) and by utilizing as primers anoligonucleotide consisting of the nucleotide sequence shown in SEQ IDNO: 394 and an oligonucleotide consisting of the nucleotide sequenceshown in SEQ ID NO: 395. The PCR utilized KOD-plus (Toyobo Company). ThePCR carried out after conducting a maintenance at 94° C. for 2 minutes;30 cycles of a cycle that included maintaining 94° C. for 30 seconds,followed by 50° C. for 30 seconds, and followed by 68° C. for 60seconds; and a final maintenance at 68° C. for 30 seconds. The amplifiedDNA was recovered and purified with MagExtractor-PCR & Gel-Clean up(Toyobo Company) by conducting the procedures according to the attachedmanual. After digesting the purified DNA with restriction enzymesEcoT221 and SacI, the DNA comprising the nucleotide sequence shown inSEQ ID NO: 214 was recovered. After digesting plasmid pUCrSt657 obtainedin Example 16(2) with restriction enzymes EcoT221 and SacI, there wasisolated a DNA of about 2.9 kbp having a nucleotide sequence derivedfrom pUC19 and a sequence encoding a chloroplast transit peptide ofsoybean (cv. Jack) RuBPC small subunit. The obtained DNA and the aboveDNA comprising the nucleotide sequence shown in SEQ ID NO: 214 wereligated to obtain pUCrSt657soy (FIG. 48) containing a chimeric DNA inwhich the present invention DNA (A1)S was connected immediately afterthe nucleotide sequence encoding the chloroplast transit peptide ofsoybean (cv. Jack) RuBPC small subunit without a change of frames in thecodons.

The obtained plasmid pUCrSt657soy was digested with restriction enzymesBamHI and SacI to isolate a DNA comprising a nucleotide sequence shownin SEQ ID NO: 214. Said DNA was inserted between the restriction enzymesite of BglII and the restriction enzyme site of SacI of plasmidpNdG6-ΔT obtained in Example 16(2) to obtain plasmidpSUM-NdG6-rSt-657soy (FIG. 49) wherein the CR16G6 promoter has connecteddownstream the chimeric DNA in which the present invention DNA (A1)S wasconnected immediately after the nucleotide sequence encoding thechloroplast transit peptide of soybean (cv. Jack) RuBPC small subunitwithout a change of frames in the codons.

Next, the plasmid was introduced into E. coli DH5α competent cells(Takara Shuzo Company) and the ampicillin resistant cells were selected.Further, the nucleotide sequences of the plasmids contained in theselected ampicillin resistant strains were determined by utilizingBigDye Terminator Cycle Sequencing Ready Reaction kit v3.0 (PE AppliedBiosystems Company) and DNA sequencer 3100 (PE Applied BiosystemsCompany). As a result, it was confirmed that plasmidpSUM-NdG6-rSt-657soy had the nucleotide sequence shown in SEQ ID NO:214.

(2) Construction of a Chloroplast Expression Plasmid Having the PresentInvention DNA (A1)S for Direct Introduction—Part (2)

A plasmid was constructed for introducing the present invention DNA(A1)S into a plant with the particle gun method. The plasmid contained achimeric DNA in which the present invention DNA (A1)S was connectedimmediately after the nucleotide sequences encoding the chloroplasttransit peptide of soybean (cv. Jack) RuBPC small subunit and encodingthereafter 12 amino acids of the mature protein, without a change offrames in the codons. First, DNA comprising the nucleotide sequenceshown in SEQ ID NO: 214 was amplified by PCR. The PCR was conducted byutilizing as a template pKSN657soy obtained in Example 46(2) and byutilizing as primers an oligonucleotide consisting of the nucleotidesequence shown in SEQ ID NO: 395 and an oligonucleotide consisting ofthe nucleotide sequence shown in SEQ ID NO: 396. The PCR utilizedKOD-plus (Toyobo Company). The PCR carried out after conducting amaintenance at 94° C. for 2 minutes; 25 cycles of a cycle that includedmaintaining 94° C. for 30 seconds, followed by 46° C. for 30 seconds,and followed by 68° C. for 60 seconds; and a final maintenance at 68° C.for 3 minutes. The amplified DNA was recovered and purified withMagExtractor-PCR & Gel-Clean up (Toyobo Company) by conducting theprocedures according to the attached manual. After digesting thepurified DNA with restriction enzyme SacI, the DNA comprising thenucleotide sequence shown in SEQ ID NO: 214 was recovered.

Plasmid pKFrSt12-657 obtained in Example 16(3) was digested withrestriction enzyme BspHI. The DNA was then blunt ended and the 5′terminus was dephosphorylated by utilizing TaKaRa BKLKit (Takara ShuzoCompany) in accordance with the attached manual. Next, after the DNA wasdigested with restriction enzyme SacI, the DNA derived from plasmidpKFrSt12 was isolated. Said DNA was ligated with the DNA which wasdigested with SacI and which comprises the nucleotide sequence shown inSEQ ID NO: 214, in order to obtain plasmid pKFrSt12-657soy (FIG. 50)containing the chimeric DNA in which the present invention DNA (A1)S wasconnected immediately after the nucleotide sequences encoding thechloroplast transit peptide of soybean (cv. Jack) RuBPC small subunitand encoding thereafter 12 amino acids of the mature protein, without achange of frames in the codons.

The obtained plasmid pKFrSt12-657soy was digested with restrictionenzymes BamHI and SacI to isolate DNA comprising the nucleotide sequenceshown in SEQ ID NO: 214. Said DNA was inserted between the restrictionenzyme site of BglII and the restriction enzyme site of SacI of plasmidpNdG6-ΔT to obtain plasmid pSUM-NdG6-rSt12-657soy (FIG. 51) wherein theCR16G6 promoter has connected downstream the chimeric DNA in which saidDNA was connected immediately after the nucleotide sequence encoding thechloroplast transit peptide of soybean (cv. Jack) RuBPC small subunitwithout a change of frames in the codons.

Next, the plasmid was introduced into E. coli DH5α competent cells(Takara Shuzo Company) and the ampicillin resistant cells were selected.Further, the nucleotide sequences of the plasmids contained in theampicillin resistant strains were determined by utilizing BigDyeTerminator Cycle Sequencing Ready Reaction kit v3.0 (PE AppliedBiosystems Company) and DNA sequencer 3100 (PE Applied BiosytemsCompany). As a result, it was confirmed that plasmidpSUM-NdG6-rSt12-657soy had the nucleotide sequence shown in SEQ ID NO:214.

(3) Introduction of the Present Invention DNA (A1)S into Soybean

The globular embryos of soybeans (cultivar: Fayette and Jack) wereprepared according to the method described in Example 17(1), other thansubstituting the vitamin source of MS medium with the vitamin source ofB5 medium (O. L. Gamborg et al., Exp. Cell Res. (1986) 50 p151).

The obtained globular embryo was transplanted into fresh somatic embryogrowth medium and cultured for 2 to 3 days. In accordance with themethod described in Example 17(2), plasmid pSUM-NdG6-rSt457soyconstructed in Example 47(1) or plasmid pSUM-NdG6-rSt) 2-657soyconstructed in Example 47(2) was introduced to said globular embryos.

(4) Selection of Somatic Embryo with Hygromycin

Selection by hygromycin of a globular embryo after the gene introductionobtained in Example 47(3) was conducted according to the methoddescribed in Example 17(3), other than substituting the vitamin sourceof MS medium with the vitamin source of B5 medium. However, after thesecond transplant, a medium to which 0.2 (w/v)% of Gelrite was added ora liquid medium to which no Gelrite was added was utilized as thesomatic embryo selection medium. In the case of the liquid medium, theculturing had 90 gentle revolutions per minute.

(5) Selection of Somatic Embryo with Compound (II)

Selection by compound (II) of a globular embryo after the geneintroduction obtained in Example 47(3) is conducted according to themethod described in Example 17(4), other than substituting the vitaminsource of MS medium with the vitamin source of B5 medium.

(6) Plant Regeneration from the Somatic Embryo, Acclimation andCultivation

In accordance with the method described in Example 17(5), the plantregeneration is conducted from the globular embryos selected in Example47(4) or 47(5). However, the agar concentration in the developmentmedium is adjusted to 0.8 (w/v)% or 1.0 (w/v)%. Further, the vitaminsource of the MS medium of the germination medium is substituted withthe vitamin source of B5 medium.

The plant with roots and developed leaves undergo the acclimation andcultivation accordingly with the method described in Example 17(6) andare harvested.

(7) Evaluation of the Resistance to Herbicidal Compound (II)

The degree of resistance against compound (II) of the regenerated plantobtained in Example 47(6) is evaluated in accordance with the methoddescribed in Example 17(4).

(8) Construction of a Chloroplast Expression Plasmid Having the PresentInvention DNA (A1)S for Agrobacterium Introduction

A plasmid for introducing the present invention DNA (A1)S into a plantwith the agrobacterium method is constructed. PlasmidpSUM-NdG6-rSt657soy was digested with restriction enzyme NotI, to obtaina chimeric DNA in which the present invention DNA (A1)S was connectedimmediately after the nucleotide sequence encoding the chloroplasttransit peptide of soybean (cv. Jack) RuBPC small subunit without achange of frames in the codons. Said DNA was inserted into the NotIrestriction site of the above binary plasmid vector pBI121S obtained inExample 18 to obtain plasmid pBI-NdG6-rSt-657soy (FIG. 52). Further,plasmid pSUM-NdG6-rSt12-657soy was digested with restriction enzymeNotI, to isolate a chimeric DNA in which the present invention DNA (A1)Swas connected immediately after the nucleotide sequences encoding thechloroplast transit peptide of soybean (cv. Jack) RuBPC small subunitand encoding thereafter 12 amino acids of the mature protein, without achange of frames in the codons. Such a DNA was inserted into the NotIrestriction site of the above binary plasmid vector pBI121S to obtainplasmid pBI-NdG6-rSt12-657soy (FIG. 53).

(9) Introduction of the Present Invention DNA (A1)S to Tobacco

The present invention DNA (A1)S was introduced into tobacco with theagrobacterium method, utilizing plasmid pBI-NdG6-rSt-657soy andpBI-NdG6-rSt12-657soy obtained in Example 47(8).

First, in accordance with the method described in Example 19, each ofthe plasmids pBI-NdG6-rSt657soy and pBI-NdG6-rSt12-657soy was introducedinto Agrobacterium tumefaciens LBA4404 (Clontech Company). Thetransgenic agrobacterium bearing pBI-NdG6-rSt-657soy orpBI-NdG6-rSt12657soy were isolated.

Next, other than culturing overnight the transgenic agrobacteriumbearing the above plasmid at 30° C. in LB liquid medium containing 25mg/l kanamycin, said agrobacterium were utilized to introduce genes intotobacco according to the method described in Example 19. There wereobtained, respectively, transgenic tobaccos which have incorporated theT-DNA region of pBI-NdG6-rSt-657soy or pBI-NdG6-rSt12-657soy.

(10) Evaluation of the Resistance Utilizing a Leaf Piece of the PresentInvention DNA (A1)S Transgenic Tobacco

Leaves were taken from 35 transgenic tobaccos obtained in Example 47(9).Each leaf was divided into pieces in which each piece was 5 to 7 mmwide. Leaf pieces were planted onto MS agar medium containing 0, 0.05,0.1 or 0.2 mg/L of compound (II) and cultured in the light at roomtemperature. On the 11th day of culturing, the herbicidal damage of eachof the leaf pieces was observed. Further, leaf pieces were planted ontoMS agar mediums containing 0, 0.01, 0.02, 0.05 or 0.1 mg/L of compound(XIII) and cultured in the light at room temperature. On the 7th day ofculturing, the herbicidal damage of each of the leaf pieces was observedAs a control, 20 leaf pieces of tobacco to which no genetic introductionhas been conducted (hereinafter, referred to as “wild type tobacco”)were utilized on each concentration. An average score for each group wasdetermined by scoring 1 point to a leaf piece that continuously grew,0.5 points to a halfly withered leaf piece in which chemical damage wasobserved, and 0 points to a leaf piece which turned white and hadwithered. The leaf pieces of the tobacco to which the present inventionDNA (A1)S (the T-DNA region of plasmid pBI-NdG6-rSt-657soy orpBI-NdG6-rSt12-657soy) has been introduced provided a higher score thanthe wild type tobacco with each of compound (II) and compound (XII).

Example 48 Obtaining the Present Invention DNA (A16)

(1) Preparation of the Chromosomal DNA of Streptomyces ornatus IFO13069t

Under the method described in Example 31(1), the chromosomal DNA ofStreptomyces ornatus IFO13069t was prepared.

(2) Isolation of DNA Having a Partial Nucleotide Sequence of the PresentInvention DNA (A11)

PCR was conducted by utilizing as the template the chromosomal DNAprepared from Streptomyces ornatus IFO 13069t in Example 48(1) and byutilizing primer pairing 14, in accordance with the method described inExample 29. Similarly to Example 31(2), the amplified DNA was clonedinto cloning vector pCRII-TOPO (Invitrogen Company). The sequencethereof was analyzed. As a result, the nucleotide sequence shown innucleotides 343 to 1069 of the nucleotide sequence shown in SEQ ID NO:225 was provided.

Further, the chromosomal DNA prepared in Example 48(1) was digested withrestriction enzyme PvuII. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 265 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizng the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 266 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1 to 501 of the nucleotide sequence shownin SEQ ID NO: 235 was provided.

Further, the chromosomal DNA prepared in Example 48(1) was digested withrestriction enzyme PvuII. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 267 and primer AP1. Next PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 268 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1044 to 1454 of the nucleotide sequenceshown in SEQ ID NO: 235 was provided.

(3) Sequence Analysis of the Present Invention DNA (A16)

The nucleotide sequence shown in SEQ ID NO: 235 was obtained byconnecting the nucleotide sequences provided by the DNA obtained inExample 48(2). Two open reading frames (ORF) were present in saidnucleotide sequence. As such, there was contained a nucleotide sequence(SEQ ID NO: 225) consisting of 1251 nucleotides (inclusive of the stopcodon) and encoding a 416 amino acid residue (SEQ ID NO: 215) and anucleotide sequence (SEQ ID NO: 255) consisting of 198 nucleotides(inclusive of the stop codon) and encoding a 65 amino acid residue (SEQID NO: 245). The molecular weight of the protein consisting of the aminoacid sequence (SEQ ID NO: 215) encoded by the nucleotide sequence shownin SEQ ID NO: 225 was calculated to be 46013 Da. Further, the molecularweight of the protein consisting of the amino acid sequence (SEQ ID NO:245) encoded by the nucleotide sequence shown in SEQ ID NO: 255 wascalculated to be 6768 Da.

Example 49 Expression of the Present Invention DNA (A16) in E. Coli

(1) Production of a Transformed E. coli having the Present Invention DNA(A16)

PCR was conducted by utilizing the GeneAmp High Fidelity PCR System(Applied Biosystems Japan Company) and by utilizing as the template thechromosomal DNA prepared from Streptomyces ornatus IFO 13069t in Example48(1). As the primers, there was utilized a pairing of theoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 269and the oligonucleotide having the nucleotide sequence shown in SEQ IDNO: 286. The PCR reaction solution amounted to 50 μl by adding the 2primers each amounting to 200 nM, 50 ng of the above chromosomal DNA,5.0 μl of dNTP mix (a mixture of 2.0 mM of each of the 4 types of dNTP;Clontech Company), 5.0 μl of 10× buffer (containing MgCl₂) and 0.5 μl ofGeneAmp HF enzyme mix and by adding distilled water. The reactionconditions of the PCR were after maintaining 97° C. for 1 minute;repeating 10 cycles of a cycle that included maintaining 97° C. for 15seconds, followed by 60° C. for 30 seconds, and followed by 72° C. for90 seconds; then conducting 15 cycles of a cycle that includedmaintaining 97° C. for 15 seconds, followed by 60° C. for 30 seconds andfollowed by 72° C. for 90 seconds (wherein 20 seconds was added to themaintenance at 72° C. for each cycle); and then maintaining 72° C. for 7minutes. Similarly to Example 32(1), the DNA was purified from thereaction solution of PCR and cloned into the cloning vector pCRII-TOPO(Invitrogen Company). The nucleotide sequence of the obtained plasmidDNA was analyzed by utilizing as primers the oligonucleotides having thenucleotide sequences shown, respectively, in SEQ ID NOs: 57, 59, 267,286 and 288. Based on the obtained results, the plasmid having thenucleotide sequence shown in SEQ ID NO: 235 was designated as pCR452F.Similarly to Example 32(1), pCR452F was digested with restrictionenzymes NdeI and HindIII. A DNA of about 1.5 kbp was purified from thedigestion products. The obtained DNA and the plasmid pKSN2 digested withNdeI and HindIII were ligated to obtain a plasmid containing thenucleotide sequence shown in SEQ ID NO: 235, in which the DNA encodingthe present invention protein (A16) is inserted between the NdeI siteand the HindIII site of pKSN2 (hereinafter referred to as “pKSN452F”).Said plasmid was introduced into E. Coli JM109. The obtained E. colitransformant was designated JM109/pKSN452F.

(2) Expression of the Present Invention Protein (A16) in E. coli andRecovery of Said Protein

Similarly to Example 4(2), each of E. coli JM109/pKSN452F andJM109/pKSN2 was cultured. The cells were recovered. Cell lysatesolutions were prepared. Under the method described in Example 4(2),supernatant fractions were prepared from the cell lysate solutions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN452F is referred to as “E. coli pKSN452F extract” and thesupernatant fraction obtained from E. coli JM109/pKSN2 is referred to as“E. coli pKSN2 extract”).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Similarly to Example 32(3), reaction solutions of 30 μl were preparedand maintained for 10 minutes at 30° C. However, as the supernatantfraction, the supernatant fraction prepared in Example 49(2) (E. colipKSN452F extract or E. coli pKSN2 extract) was utilized. The reactionsolutions after the maintenance were extracted with ethyl acetate andthe extracted layers were TLC analyzed. After developing the TLC plate,the presence of a spot thereon corresponding to compound (III) labeledwith ¹⁴C were examined (Rf value 0.24 and 0.29). A spot corresponding tocompound (III) was detected from the reaction solution containing E.coli pKSN452F extract. In contrast, such a spot was not detected fromthe reaction solution containing E. coli pKSN2 extract.

Example 50 Obtaining the Present Invention DNA (A17)

(1) Preparation of the Chromosomal DNA of Streptomyces griseus ATCC10137

Under the method described in Example 31(1), the chromosomal DNA ofStreptomyces griseus ATCC 10137 was prepared.

(2) Isolation of DNA Having a Partial Nucleotide Sequence of the PresentInvention DNA (A17)

PCR was conducted by utilizing as the template the chromosomal DNA ofStreptomyces griseus ATCC 10137 prepared in Example 50(1) and byutilizing primer pairing 14, in accordance with the method described inExample 29. Similarly to Example 31(2), the amplified DNA was cloned tocloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequencethereof was analyzed. As a result, the nucleotide sequence shown innucleotides 343 to 1069 of the nucleotide sequence shown in SEQ ID NO:226 was provided.

Further, the chromosomal DNA prepared in Example 50(1) was digested withrestriction enzyme SmaI. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 270 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 271 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1 to 361 of the nucleotide sequence shownin SEQ ID NO: 236 was provided.

Further, the chromosomal DNA prepared in Example 50(1) was digested withrestriction enzyme PvuII. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 272 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 273 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1035 to 1454 of the nucleotide sequenceshown in SEQ ID NO: 236 was provided.

(3) Sequence Analysis of the Present Invention DNA (A17)

The nucleotide sequence shown in SEQ ID NO: 236 was obtained byconnecting the nucleotide sequences provided by the DNA obtained inExample 50(2). Two open reading frames (ORF) were present in saidnucleotide sequence. As such, there was contained a nucleotide sequence(SEQ ID NO: 226) consisting of 1251 nucleotides (inclusive of the stopcodon) and encoding a 416 amino acid residue (SEQ ID NO: 216) and anucleotide sequence (SEQ ID NO: 256) consisting of 198 nucleotides(inclusive of the stop codon) and encoding a 65 amino acid residue (SEQID NO: 246). The molecular weight of the protein consisting of the aminoacid sequence (SEQ ID NO: 216) encoded by the nucleotide sequence shownin SEQ ID NO: 226 was calculated to be 46082 Da. The molecular weight ofte protein consisting of the amino acid sequence (SEQ ID NO: 246)encoded by the nucleotide sequence shown in SEQ ID NO: 256 wascalculated to be 6768 Da. The nucleotide sequence shown in SEQ ID NO:256 is 100% identical to the nucleotide sequence shown in SEQ ID NO:255. The amino acid sequence shown in SEQ ID NO: 246 is 100% identicalto the amino acid sequence shown in SEQ ID NO: 245.

Example 51 Expression of the Present Invention DNA (A17) in E. Coli

(1) Production of a Transformed E. coli Having the Present Invention DNA(A17)

PCR was conducted similarly to Example 32(1), other than utilizing as atemplate the chromosomal DNA prepared from Streptomyces griseus ATCC10137 in Example 50(1) and utilizing as the primers the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO; 274 and anoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 275.Similarly to Example 32(1), the DNA was purified from the reactionsolution of PCR and cloned into the cloning vector pCRII-TOPO(Invitrogen Company). The nucleotide sequence of the obtained plasmidDNA was sequenced by utilizing as primers the oligonucleotides havingthe nucleotide sequences shown, respectively, in SEQ ID NOs: 57, 59,274, 276 and 277. Based on the obtained results, the plasmid having thenucleotide sequence shown in SEQ ID NO: 236 was designated as pCR608F.Similarly to Example 32(1), pCR608F was digested with restrictionenzymes NdeI and HindIII. A DNA of about 1.5 kbp was purified from thedigestion products. The obtained DNA and the plasmid pKSN2 digested withNdeI and HindIII were ligated to obtain a plasmid containing thenucleotide sequence shown in SEQ ID NO: 236, in which the DNA encodingthe present invention protein (A17) is inserted between the NdeI siteand the HindIII site of pKSN2 (hereinafter referred to as “pKSN608F”).Said plasmid was introduced into E. Coli JM109. The obtained E. colitransformant was designated JM109/pKSN608F.

(2) Expression of the Present Invention Protein (A17) in E. coli andRecovery of Said Protein

Similarly to Example 4(2), each of E. coli JM109/pKSN60SF andJM109/pKSN2 was cultured. The cells were recovered. Cell lysatesolutions were prepared. Under the method described in Example 4(2),supernatant fractions were prepared from the cell lysate solutions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN60SF is referred to as “E. coli pKSN608F extract” and thesupernatant fraction obtained from E. coli JM109/pKSN2 is referred to as“E. coli pKSN2 extract”).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Similarly to Example 32(3), reaction solutions of 30 μl were preparedand maintained for 10 minutes at 30° C. However, as the supernatantfraction, the supernatant fraction prepared in Example 51(2) (E. colipKSN608F extract or E. coli pKSN2 extract) was utilized. The reactionsolutions after the maintenance were extracted with ethyl acetate andthe extracted layers were TLC analyzed. After developing the TLC plate,the presence of a spot thereon corresponding to compound (III) labeledwith ¹⁴C were examined (Rf value 0.24 and 0.29). A spot corresponding tocompound (III) was detected from the reaction solution containing E.coli pKSN608F extract. In contrast, such a spot was not detected fromthe reaction solution containing E. coli pKSN2 extract.

Example 52 Obtaining the Present Invention DNA (A18)

(1) Preparation of the Chromosomal DNA of Streptomyces achromogenes IFO12735

Under the method described in Example 31(1), the chromosomal DNA ofStreptomyces achromogenes IFO 12735 was prepared.

2) Isolation of DNA Having a Partial Nucleotide Sequence of the PresentInvention DNA (A18)

PCR was conducted by utilizing as the template the chromosomal DNA ofStreptomyces achromogenes IFO 12735 prepared in Example 52(1) and byutilizing primer pairing 17, in accordance with the method described inExample 29. Similarly to Example 31(2), the amplified DNA was cloned tocloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequencethereof was analyzed. As a result, the nucleotide sequence shown innucleotides 526 to 1048 of the nucleotide sequence shown in SEQ ID NO:227 was provided.

Further, the chromosomal DNA prepared in Example 52(1) was digested withrestriction enzyme HincII. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the fit PCR products, by utilizing the obtained library as thetemplate and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 278 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 279 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1 to 600 of the nucleotide sequence shownin SEQ ID NO: 237 was provided.

Further, the chromosomal DNA prepared in Example 52(1) was digested withrestriction enzyme Ball. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 163 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 164 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 983 to 1449 of the nucleotide sequenceshown in SEQ ID NO: 237 was provided.

(3) Sequence Analysis of the Present Invention DNA (A18)

The nucleotide sequence shown in SEQ ID NO: 237 was obtained byconnecting the nucleotide sequences provided by the DNA obtained inExample 52(2). Two open reading frames (ORF) were present in saidnucleotide sequence. As such, there was contained a nucleotide sequence(SEQ ID NO: 227) consisting of 1230 nucleotides (inclusive of the stopcodon) and encoding a 409 amino acid residue (SEQ ID NO: 217) and anucleotide sequence (SEQ ID NO: 257) consisting of 207 nucleotides(inclusive of the stop codon) and encoding a 68 amino acid residue (SEQID NO: 247). The molecular weight of the protein consisting of the aminoacid sequence (SEQ ID NO: 217) encoded by the nucleotide sequence shownin SEQ ID NO: 227 was calculated to be 45099 Da. The molecular weight ofthe protein consisting of the amino acid sequence (SEQ ID NO: 247)encoded by te nucleotide sequence shown in SEQ ID NO: 257 was calculatedto be 7193 Da.

Example 53 Expression of the Present Invention DNA (A18) in E. Coli

(1) Production of a Transformed E. coli Having the Present Invention DNA(A18)

PCR was conducted similarly to Example 49(1), other than utilizing as atemplate the chromosomal DNA prepared from Streptomyces achromogenes IFO12735 in Example 52(1) and utilizing as the primers the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 183 and anoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 280.Similarly to Example 32(1), the DNA was purified from the reactionsolution of PCR and cloned into the cloning vector pCRII-TOPO(Invitrogen Company). The nucleotide sequence of the obtained plasmidDNA was analyzed by utilizing as primers the oligonucleotides having thenucleotide sequences shown, respectively, in SEQ ID NOs: 67, 68, 163,279 and 281. Based on the obtained results, the plasmid having thenucleotide sequence shown in SEQ ID NO: 237 was designated as pCR646BF.Similarly to Example 32(1), pCR646BF was digested with restrictionenzymes NdeI and HindIII. A DNA of about 1.5 kbp was purified from thedigestion products. The obtained DNA and the plasmid pKSN2 digested withNdeI and HindIII were ligated to obtain a plasmid containing thenucleotide sequence shown in SEQ ID NO; 237, in which the DNA encodingthe present invention protein (A18) is inserted between the NdeI siteand the HindIII site of pKSN2 (hereinafter referred to as “pKSN646BF”).Said plasmid was introduced into E. Coli JM109. The obtained E. colitransformant was designated JM109/pKSN646BF.

(2) Expression of the Present Invention Protein (A18) in E. coli andRecovery of said Protein

Similarly to Example 4(2), each of E. coli JM109/pKSN464BF andJM109/pKSN2 was cultured. The cells were recovered. Cell lysatesolutions were prepared. Under the method described in Example 4(2),supernatant fractions were prepared from the cell lysate solutions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN646BF is referred to as “E. coli pKSN646BF extract” and thesupernatant fraction obtained from E. coli JM109/pKSN2 is referred to as“E. coli pKSN2 extract”).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Similarly to Example 32(3), reaction solutions of 30 μl were preparedand maintained for 10 minutes at 30° C. However, as the supernatantfraction, the supernatant fraction prepared in Example 53(2) (E. colipKSN646BF extract or E. coli pKSN2 extract) was utilized. The reactionsolutions after the maintenance were extracted with ethyl acetate andthe extracted layers were TLC analyzed. After developing the TLC plate,the presence of a spot thereon corresponding to compound (III) labeledwith ¹⁴C were examined (Rf value 0.24 and 0.29). A spot corresponding tocompound (III) was detected from the reaction solution containing E.coli pKSN646BF extract. In contrast, such a spot was not detected fromthe reaction solution containing E. coli pKSN2 extract.

Example 54 Obtaining the Present Invention DNA (A19)

(1) Preparation of the Chromosomal DNA of Streptomyces griseus IFO13849T

Under the method described in Example 31(1), the chromosomal DNA ofStreptomyces griseus IFO 13849T was prepared.

(2) Isolation of DNA Having a Partial Nucleotide Sequence of the PresentInvention DNA (A19)

PCR was conducted by utilizing as the template the chromosomal DNA ofStreptomyces griseus IFO 13849T prepared in Example 54(1) and byutilizing primer pairing 14, in accordance with the method described inExample 29. Similarly to Example 31(2), the amplified DNA was cloned tocloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequencethereof was analyzed. As a result the nucleotide sequence shown innucleotides 343 to 1069 of the nucleotide sequence shown in SEQ ID NO:228 was provided.

Further, the chromosomal DNA prepared in Example 54(1) was digested withrestriction enzyme SmaI. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO., 282 and primer AP1. Next, PCR wasconducted under the conditions described in Example 26(3), by utilizingthe first PCR products as the template and by utilizing theoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 283and primer AP2. The nucleotide sequence of the obtained DNA wasanalyzed. The nucleotide sequence shown in nucleotides 1 to 358 of thenucleotide sequence shown in SEQ ID NO: 238 was provided.

Further, the chromosomal DNA prepared in Example 54(1) was digested withrestriction enzyme HindIII. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCP products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 284 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 285 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1005 to 1454 of the nucleotide sequenceshown in SEQ ID NO: 238 was provided.

(3) Sequence Analysis of the Present Invention DNA (A19)

The nucleotide sequence shown in SEQ ID NO: 238 was obtained byconnecting the nucleotide sequences provided by the DNA obtained inExample 54(2). Two open reading frames (ORF) were present in saidnucleotide sequence. As such, there was contained a nucleotide sequence(SEQ ID NO: 228) consisting of 1251 nucleotides (inclusive of the stopcodon) and encoding a 416 amino acid residue (SEQ ID NO: 218) and anucleotide sequence (SEQ ID NO: 258) consisting of 156 nucleotides(inclusive of the stop codon) and encoding a 51 amino acid residue (SEQID NO: 248). The molecular weight of the protein consisting of the aminoacid sequence (SEQ ID NO: 218) encoded by the nucleotide sequence shownin SEQ ID NO: 228 was calculated to be 45903 Da. The molecular weight ofthe protein consisting of the amino acid sequence (SEQ ID NO 248)encoded by the nucleotide sequence shown in SEQ ID NO: 258 wascalculated to be 5175 Da.

Example 55 Expression of the Present Invention DNA (A19) in E. Coli

(1) Production of a Transformed E. coli Having the Present Invention DNA(A19)

PCR was conducted similarly to Example 49(1), other than utilizing as atemplate the chromosomal DNA prepared from Streptomyces griseus IFO13849T in Example 54(1) and utilizing as the primers the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 286 and anoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 287.Similarly to Example 32(1), the DNA was purified from the reactionsolution of PCR and cloned into the cloning vector pCRII-TOPO(Invitrogen Company). The nucleotide sequence of the obtained plasmidDNA was analyzed by utilizing as primers the oligonucleotides having thenucleotide sequences shown, respectively, in SEQ ID NOs: 57, 59, 284,286 and 288. Based on the obtained results, the plasmid having thenucleotide sequence shown in SEQ ID NO: 238 was designated as pCR1502F.Similarly to Example 32(1), pCR1502F was digested with restrictionenzymes NdeI and HindIII. A DNA of about 1.5 kbp was purified from thedigestion products. The obtained DNA and the plasmid pKSN2 digested withNdeI and HindIII were ligated to obtain a plasmid containing thenucleotide sequence shown in SEQ ID NO: 238, in which the DNA encodingthe present invention protein (A19) is inserted between the NdeI siteand the HindIII site of pKSN2 (hereinafter referred to as “pKSN1502F”).Said plasmid was introduced into E. Coli JM109. The obtained E. colitransformant was designated JM109/pKSN1502F.

(2) Expression of the Present Invention Protein (A18) in E. coli andRecovery of Said Protein

Similarly to Example 4(2), each of E. coli JM109/pKSN1502F andJM109/pKSN2 was cultured. The cells were recovered. Cell lysatesolutions were prepared. Under the method described in Example 4(2),supernatant fractions were prepared from the cell lysate solutions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN1502F is referred to as “E. coli pKSN1502F extract” and thesupernatant fraction obtained from E. coli JM109/pKSN2 is referred to as“E. coli pKSN2 extract”).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Similarly to Example 32(3), reaction solutions of 30 μl were preparedand maintained for 10 minutes at 30° C. However, as the supernatantfraction, the supernatant fraction prepared in Example 55(2) (E. colipKSN1502F extract or E. coli pKSN2 extract) was utilized. The reactionsolutions after the maintenance were extracted with ethyl acetate andthe extracted layers were TLC analyzed. After developing the TLC plate,the presence of a spot thereon corresponding to compound (III) labeledwith ¹⁴C were examined (Rf value 0.24 and 0.29). A spot corresponding tocompound (III) was detected from the reaction solution containing E.coli pKSN1502F extract. In contrast, such a spot was not detected fromthe reaction solution containing E. coli pKSN2 extract.

Example 56 Obtaining the Present Invention DNA (A20)

(1) Preparation of the Chromosomal DNA of Streptomyces lanatus IFO12787T

Under the method described in Example 31(1), the chromosomal DNA ofStreptomyces lanatus IFO 12787T was prepared.

(2) Isolation of DNA Having a Partial Nucleotide Sequence of the PresentInvention DNA (A20)

PCR was conducted by utilizing as the template the chromosomal DNA ofStreptomyces lanatus IFO 12787T prepared in Example 56(1) and byutilizing primer pairing 14, in accordance with the method described inExample 29. Similarly to Example 31(2), the amplified DNA was cloned tocloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequencethereof was analyzed. As a result, the nucleotide sequence shown innucleotides 304 to 1036 of the nucleotide sequence shown in SEQ ID NO:229 was provided.

Further, the chromosomal DNA prepared in Example 56(1) was digested withrestriction enzyme PmacI. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 278 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 289 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1 to 318 of the nucleotide sequence shownin SEQ ID NO: 239 was provided.

Further, the chromosomal DNA prepared in Example 56(1) was digested withrestriction enzyme StuI. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 290 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 291 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 969 to 1461 of the nucleotide sequenceshown in SEQ ID NO: 239 was provided.

(3) Sequence Analysis of the Present Invention DNA (A20)

The nucleotide sequence shown in SEQ ID NO: 239 was obtained byconnecting the nucleotide sequences provided by the DNA obtained inExample 56(2). Two open reading frames (ORF) were present in saidnucleotide sequence. As such, there was contained a nucleotide sequence(SEQ ID NO: 229) consisting of 1218 nucleotides (inclusive of the stopcodon) and encoding a 405 amino acid residue (SEQ ID NO: 219) and anucleotide sequence (SEQ ID NO: 259) consisting of 231 nucleotides(inclusive of the stop codon) and encoding a 76 amino acid residue (SEQID NO: 249). The molecular weight of the protein consisting of the aminoacid sequence (SEQ ID NO: 219) encoded by the nucleotide sequence shownin SEQ ID NO: 229 was calculated to be 45071 Da. The molecular weight ofthe protein consisting of the amino acid sequence (SEQ ID NO: 249)encoded by the nucleotide sequence shown in SEQ ID NO: 259 wascalculated to be 7816 Da.

Example 57 Expression of the Present Invention DNA (A20) in E. Coli

(1) Production of a Transformed E. coli Having the Present Invention DNA(A20)

PCR was conducted similarly to Example 49(1), other than utilizing as atemplate the chromosomal DNA prepared from Streptomyces lanatus IFO12787T in Example 56(1) and utilizing as the primers the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 292 and anoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 293.Similarly to Example 32(1), the DNA was purified from the reactionsolution of PCR and cloned into the cloning vector pCRII-TOPO(Invitrogen Company). The nucleotide sequence of the obtained plasmidDNA was analyzed by utilizing as primers the oligonucleotides having thenucleotide sequences shown, respectively, in SEQ ID NOs: 67, 68, 188,278 and 290. Based on the obtained results, the plasmid having thenucleotide sequence shown in SEQ ID NO: 239 was designated as pCR1525F.Similarly to Example 32(1), pCR1525F was digested with restrictionenzymes NdeI and HindIII. A DNA of about 1.5 kbp was purified from thedigestion products. The obtained DNA and the plasmid pKSN2 digested withNdeI and HindIII were ligated to obtain a plasmid containing thenucleotide sequence shown in SEQ ID NO: 239, in which the DNA encodingthe present invention protein (A20) is inserted between the NdeI siteand the HindIII site of pKSN2 (hereinafter referred to as “pKSN1525F”).Said plasmid was introduced into E. Coli JM109. The obtained E. colitransformant was designated JM109/pKSN1525F.

(2) Expression of the Present Invention Protein (A20) in E. coli andRecovery of Said Protein

Similarly to Example 4(2), each of E. coli JM109/pKSN1525F andJM109/pKSN2 was cultured. The cells were recovered. Cell lysatesolutions were prepared. Under the method described in Example 4(2),supernatant fractions were prepared from the cell lysate solutions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN1525F is referred to as “E. coli pKSN1525F extract” and thesupernatant fraction obtained from E. coli JM109pKSN2 is referred to as“E. coli pKSN2 extract”).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Similarly to Example 32(3), reaction solutions of 30 μl were preparedand maintained for 10 minutes at 30° C. However, as the supernatantfraction, the supernatant fraction prepared in Example 57(2) (E. colipKSN1525F extract or E. coli pKSN2 extract) was utilized. The reactionsolutions after the maintenance were extracted with ethyl acetate andthe extracted layers were TLC analyzed. After developing the TLC plate,the presence of a spot thereon corresponding to compound (III) labeledwith ¹⁴C were examined (Rf value 0.24 and 0.29). A spot corresponding tocompound (III) was detected from the reaction solution containing E.coli pKSN1525F extract. In contrast, such a spot was not detected fromthe reaction solution containing E. coli pKSN2 extract.

Example 58 Obtaining the Present Invention DNA (A21)

(1) Preparation of the Chromosomal DNA of Streptomyces misawanensis IFO13855T

Under the method described in Example 31(1), the chromosomal DNA ofStreptomyces misawanensis IFO 13855T was prepared.

(2) Isolation of DNA Having a Partial Nucleotide Sequence of the PresentInvention DNA (A21)

PCR was conducted by utilizing as the template the chromosomal DNA ofStreptomyces misawanensis IFO 13855T prepared in Example 58(1) and byutilizing primer pairing 14, in accordance with the method described inExample 29. Similarly to Example 31(2), the amplified DNA was cloned tocloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequencethereof was analyzed. As a result, the nucleotide sequence shown innucleotides 328 to 1063 of the nucleotide sequence shown in SEQ ID NO:230 was provided.

Further, the chromosomal DNA prepared in Example 58(1) was digested withrestriction enzyme SmaI. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 294 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 295 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1 to 341 of the nucleotide sequence shownSEQ ID NO: 240 was provided.

Further, the chromosomal DNA prepared in Example 58(1) was digested withrestriction enzyme HincII. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 296 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 297 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1017 to 1458 of the nucleotide sequenceshown in SEQ ID NO: 240 was provided.

(3) Sequence Analysis of the Present Invention DNA (A21)

The nucleotide sequence shown in SEQ ID NO: 240 was obtained byconnecting the nucleotide sequences provided by the DNA obtained inExample 58(2). Two open reading frames (ORF) were present in saidnucleotide sequence. As such, there was contained a nucleotide sequence(SEQ ID NO: 230) consisting of 1245 nucleotides (inclusive of the stopcodon) and encoding a 414 amino acid residue (SEQ ID NO: 220) and anucleotide sequence (SEQ ID NO: 260) consisting of 201 nucleotides(inclusive of the stop codon) And encoding a 66 amino acid residue (SEQID NO: 250). The molecular weight of the protein consisting of the aminoacid sequence (SEQ ID NO: 220) encoded by the nucleotide sequence shownin SEQ ID NO: 230 was calculated to be 45806 Da. The molecular weight ofthe protein consisting of the amino acid sequence (SEQ ID NO: 250)encoded by the nucleotide sequence shown in SEQ ID NO: 260 wascalculated to be 6712 Da.

Example 59 Expression of the Present Invention DNA (A21) in E. Coli

(1) Production of a Transformed E. coli Having the Present Invention DNA(A21)

PCR was conducted similarly to Example 32(1), other than utilizing as atemplate the chromosomal DNA prepared from Streptomyces misawanensis IFO13855T in Example 58(1) and utilizing as the primers the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 298 and anoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 299.Similarly to Example 32(1), the DNA was purified from the reactionsolution of PCR and cloned into the cloning vector pCRII-TOPO(Invitrogen Company). The nucleotide sequence of the obtained plasmidDNA was analyzed by utilizing as primers the oligonucleotides having thenucleotide sequences shown, respectively, in SEQ ID NOs: 57, 59, 296,298 and 300. Based on the obtained results, the plasmid having thenucleotide sequence shown in SEQ ID NO: 240 was designated as pCR1543BF.Similarly to Example 32(1), pCR1543BF was digested with restrictionenzymes NdeI and HindIII. A DNA of about 1.5 kbp was purified from thedigestion products. The obtained DNA and the plasmid pKSN2 digested withNdeI and HindIII were ligated to obtain a plasmid containing thenucleotide sequence shown in SEQ ID NO: 240, in which the DNA encodingthe present invention protein (A21) is inserted between the NdeI siteand the HindIII site of pKSN2 (hereinafter referred to as“pKSN154313F”). Said plasmid was introduced into E. Coli JM109. Theobtained E. coli transformant was designated JM109/pKSN15439F.

(2) Expression of the Present Invention Protein (A21) in E. coli andRecovery of Said Protein

Similarly to Example 4(2), each of E. coli JM109/pKSN1543BF andJM109/pKSN2 was cultured. The cells were recovered. Cell lysatesolutions were prepared. Under the method described in Example 4(2),supernatant fractions were prepared from the cell lysate solutions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN1543BF is referred to as “E. coli pKSN1543BF extract” and thesupernatant fraction obtained from E. coli JM109/pKSN2 is referred to as“E. coli pKSN2 extract”).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Similarly to Example 32(3), reaction solutions of 3011 were prepared andmaintained for 10 minutes at 30° C. However, as the supernatantfraction, the supernatant fraction prepared in Example 59(2) (E. colipKSN1543BF extract or E. coli pKSN2 extract) was utilized. The reactionsolutions after the maintenance were extracted with ethyl acetate andthe extracted layers were TLC analyzed. After developing the TLC plate,the presence of a spot thereon corresponding to compound (III) labeledwith ¹⁴C were examined (Rf value 0.24 and 0.29). A spot corresponding tocompound (III) was detected from the reaction solution containing E.coli pKSN1543BF extract. In contrast, such a spot was not detected fromthe reaction solution containing E. coli pKSN2 extract.

Example 60 Obtaining the Present Invention DNA (A12)

(1) Preparation of the Chromosomal DNA of Streptomyces pallidus IFO13434T

Under the method described in Example 31(1), the chromosomal DNA ofStreptomyces pallidus IFO 13434T was prepared.

(2) Isolation of DNA Having a Partial Nucleotide Sequence of the PresentInvention DNA (A22)

PCR was conducted by utilizing as the template the chromosomal DNA ofStreptomyces pallidus IFO 13434T prepared in Example 60(1) and byutilizing primer pairing 15, in accordance with the method described inExample 29. Similarly to Example 31(2), the amplified DNA was cloned tocloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequencethereof was analyzed. As a result, the nucleotide sequence shown innucleotides 483 to 1048 of the nucleotide sequence shown in SEQ ID NO:231 was provided.

Further, the chromosomal DNA prepared in Example 60(1) was digested withrestriction enzyme SmaI. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the fist PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 301 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 302 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 68 to 516 of the nucleotide sequence shownin SEQ ID NO: 241 was provided.

Further, the chromosomal DNA prepared in Example 60(1) was digested withrestriction enzyme HincII. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 302 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 303 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1 to 270 of the nucleotide sequence shownin SEQ ID NO: 241 was provided.

Further, the chromosomal DNA prepared in Example 60(1) was digested withrestriction enzyme HincII. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO; 304 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 305 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 982 to 1448 of the nucleotide sequenceshown in SEQ ID NO: 241 was provided.

(3) Sequence Analysis of the Present Invention DNA (A22)

The nucleotide sequence shown in SEQ ID NO: 241 was obtained byconnecting the nucleotide sequences provided by the DNA obtained inExample 60(2). Two open reading frames (ORF) were present in saidnucleotide sequence. As such, there was contained a nucleotide sequence(SEQ ID NO: 231) consisting of 1230 nucleotides (inclusive of the stopcodon) and encoding a 409 amino acid residue (SEQ ID NO: 221) and anucleotide sequence (SEQ ID NO: 261) consisting of 195 nucleotides(inclusive of the stop codon) and encoding a 64 amino acid residue (SEQID NO: 251). The molecular weight of the protein consisting of the aminoacid sequence (SEQ ID NO: 221) encoded by the nucleotide sequence shownin SEQ ID NO: 231 was calculated to be 45050 Da. The molecular weight ofthe protein consisting of the amino acid sequence (SEQ ID NO: 251)encoded by the nucleotide sequence shown in SEQ ID NO: 261 wascalculated to be 6914 Da.

Example 61 Expression of the Present Invention DNA (A22) in E. Coli

(1) Production of a Transformed E. coli Having the Present Invention DNA(A22)

PCR was conducted similarly to Example 32(1), other than utilizing as atemplate the chromosomal DNA prepared from Streptomyces pallidus IFO13434T in Example 60(1) and utilizing as the primers the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 306 and anoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 307.Similarly to Example 32(1), the DNA was purified from the reactionsolution of PCR and cloned into the cloning vector pCRII-TOPO(Invitrogen Company). The nucleotide sequence of the obtained plasmidDNA was analyzed by utilizing as primers the oligonucleotides having thenucleotide sequences shown, respectively, in SEQ ID NOs: 67, 68 and 308.Based on the obtained results, the plasmid having the nucleotidesequence shown in SEQ ID NO: 241 was designated as pCR1558BF. Similarlyto Example 32(1), pCR1558BF was digested with restriction enzymes NdeIand HindIII. A DNA of about 1.5 kbp was purified from the digestionproducts. The obtained DNA and the plasmid pKSN2 digested with NdeI andHindIII were ligated to obtain a plasmid containing the nucleotidesequence shown in SEQ ID NO: 241, in which the DNA encoding the presentinvention protein (A22) is inserted between the NdeI site and theHindIII site of pKSN2 (hereinafter referred to as “pKSN1558BF”). Saidplasmid was introduced into E. Coli JM109. The obtained E. colitransformant was designated JM109/pKSN1558BF.

(2) Expression of the Present Invention Protein (A22) in E. coli andRecovery of Said Protein

Similarly to Example 4(2), each of E. coli JM109/pKSN1558BF andJM109/pKSN2 was cultured. The cells were recovered. Cell lysatesolutions were prepared. Under the method described in Example 4(2),supernatant fractions were prepared from the cell lysate solutions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN1558BF is referred to as “E. coli pKSN1558BF extract” and thesupernatant fraction obtained from E. coli JM109/pKSN2 is referred to as“E. coli pKSN2 extract”).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Similarly to Example 32(3), reaction solutions of 30 μl were preparedand maintained for 10 minutes at 30° C. However, as the supernatantfraction, the supernatant fraction prepared in Example 61(2) (E. colipKSN1558BF extract or E. coli pKSN2 extract) was utilized. The reactionsolutions after the maintenance were extracted with ethyl acetate andthe extracted layers were TLC analyzed. After developing the TLC plate,the presence of a spot thereon corresponding to compound (III) labeledwith ¹⁴C were examined (Rf value 0.24 and 0.29). A spot corresponding tocompound (II) was detected form the reaction solution containing E colipKSN1558BF extract. In contrast, such a spot was not detected from thereaction solution containing E. coli pKSN2 extract.

Example 62 Obtaining the Present Invention DNA (A23)

(1) Preparation of the Chromosomal DNA of Streptomyces roseorubens IFO13682T

Under the method described in Example 31(1), the chromosomal DNA ofStreptomyces roseorubens IFO 13682T was prepared.

(2) Isolation of DNA Having a Partial Nucleotide Sequence of the PresentInvention DNA (A23)

PCR was conducted by utilizing as the template the chromosomal DNA ofStreptomyces roseorubens IFO 13682T prepared in Example 62(1) and byutilizing primer pairing 14, in accordance with the method described inExample 29. Similarly to Example 31(2), the amplified DNA was cloned tocloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequencethereof was analyzed. As a result, the nucleotide sequence shown innucleotides 289 to 1015 of the nucleotide sequence shown in SEQ ID NO:232 was provided.

Further, the chromosomal DNA prepared in Example 62(1) was digested withrestriction enzyme SmaI. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 309 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 310 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1 to 354 of the nucleotide sequence shownin SEQ ID NO: 242 was provided.

Further, the chromosomal DNA prepared in Example 62(1) was digested withrestriction enzyme PvuII. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 311 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 312 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 966 to 1411 of the nucleotide sequenceshown in SEQ ID NO: 242 was provided.

(3) Sequence Analysis of the Present Invention DNA (A23)

The nucleotide sequence shown in SEQ ID NO: 242 was obtained byconnecting the nucleotide sequences provided by the DNA obtained inExample 62(2). Two open reading frames (ORF) were present in saidnucleotide sequence. As such, there was contained a nucleotide sequence(SEQ ID NO: 232) consisting of 1197 nucleotides (inclusive of the stopcodon) and encoding a 398 amino acid residue (SEQ ID NO: 222) and anucleotide sequence (SEQ ID NO: 262) consisting of 201 nucleotides(inclusive of the stop codon) and encoding a 66 amino acid residue (SEQID NO: 252). The molecular weight of the protein consisting of the aminoacid sequence (SEQ ID NO: 222) encoded by the nucleotide sequence shownin SEQ ID NO: 232 was calculated to be 43624 Da. The molecular weight ofthe protein consisting of the amino acid sequence (SEQ ID NO: 252)encoded by the nucleotide sequence shown in SEQ ID NO: 262 wascalculated to be 6797 Da.

Example 63 Expression of the Present Invention DNA (A23) in E. Coli

(1) Production of a Transformed E. coli Having the Present Invention DNA(A23)

PCR was conducted similarly to Example 49(1), other than utilizing as atemplate the chromosomal DNA prepared from Streptomyces roseorubens IFO13682T in Example 62(1) and utilizing as the primers the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 313 and anoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 314.Similarly to Example 32(1), the DNA was purified from the reactionsolution of PCR and cloned into the cloning vector pCRII-TOPO(Invitrogen Company). The nucleotide sequence of the obtained plasmidDNA was analyzed by utilizing as primers the oligonucleotides having thenucleotide sequences shown, respectively, in SEQ ID NOs: 67, 68, 309,311 and 315. Based on the obtained results, the plasmid having thenucleotide sequence shown in SEQ ID NO: 242 was designated as pCR1584F.Similarly to Example 32(1), pCR1584F was digested with restrictionenzymes NdeI and HindIII. A DNA of about 1.5 kbp was purified from thedigestion products. The obtained DNA and the plasmid pKSN2 digested withNdeI and HindIII were ligated to obtain a plasmid containing thenucleotide sequence shown in SEQ ID NO: 242, in which the DNA encodingthe present invention protein (A23) is inserted between the NdeI siteand the HindIII site of pKSN2 (hereinafter referred to as “pKSN1584F”).Said plasmid was introduced into E. Coli JM109. The obtained E colitransformant was designated JM109/pKSN1584F.

(2) Expression of the Present Invention Protein (A23) in E. coli andRecovery of Said Protein

Similarly to Example 4(2), each of B. coli JM109/pKSN1584F andJM109/pKSN2 was cultured. The cells were recovered. Cell lysatesolutions were prepared. Under the method described in Example 4(2),supernatant fractions were prepared from the cell lysate solutions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN1584F is referred to as “E. coli pKSN1584F extract” and thesupernatant fraction obtained from E. coli JM109/pKSN2 is referred to as“E. coli pKSN2 extract”).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Similarly to Example 32(3), reaction solutions of 30 μl were preparedand maintained for 10 minutes at 30° C. However, as the supernatantfraction, the supernatant fraction prepared in Example 63(2) (E. colipKSN1584F extract or E. coli pKSN2 extract) was utilized. The reactionsolutions after the maintenance were extracted with ethyl acetate andthe extracted layers were TLC analyzed. After developing the TLC plate,the presence of a spot thereon corresponding to compound (III) labeledwith ¹⁴C were examined (Rf value 0.24 and 0.29). A spot corresponding tocompound (III) was detected from the reaction solution containing E.coli pKSN1584F extract. In contrast, such a spot was not detected fromthe reaction solution containing E. coli pKSN2 extract.

Example 64 Obtaining the Present Invention DNA (A24)

(1) Preparation of the Chromosomal DNA of Streptomyces rutgersensis IFO15875T

Under the method described in Example 31 (1), the chromosomal DNA ofStreptomyces rutgersensis IFO 15875T was prepared.

(2) Isolation of DNA Having a Partial Nucleotide Sequence of the PresentInvention DNA (A24)

PCR was conducted by utilizing as the template the chromosomal DNA ofStreptomyces rutgersensis IFO 15875T prepared in Example 64(1) and byutilizing primer pairing 14, in accordance with the method described inExample 29. Similarly to Example 31(2), the amplified DNA was cloned tocloning vector pCRII-TOPO (Invitrogen Company). The nucleotide sequencethereof was analyzed. As a result, the nucleotide sequence shown innucleotides 322 to 1057 of the nucleotide sequence shown in SEQ ID NO:233 was provided.

Further, the chromosomal DNA prepared in Example 64(1) was digested withrestriction enzyme SmaI. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 316 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 317 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1 to 384 of the nucleotide sequence shownin SEQ ID NO: 243 was provided.

Further, the chromosomal DNA prepared in Example 64(1) was digested withrestriction enzyme NaeI. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 318 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 319 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 992 to 1466 of the nucleotide sequenceshown in SEQ ID NO: 243 was provided.

(3) Sequence Analysis of the Present Invention DNA (A24)

The nucleotide sequence shown in SEQ ID NO: 243 was obtained byconnecting the nucleotide sequences provided by the DNA obtained inExample 64(2). Two open reading frames (ORF) were present in saidnucleotide sequence. As such, there was contained a nucleotide sequence(SEQ ID NO: 233) consisting of 1245 nucleotides (inclusive of the stopcodon) and encoding a 414 amino acid residue (SEQ ID NO: 223) and anucleotide sequence (SEQ ID NO: 263) consisting of 198 nucleotides(inclusive of the stop codon) and encoding a 65 amino acid residue (SEQID NO: 253). The molecular weight of the protein consisting of the aminoacid sequence (SEQ ID NO: 223) encoded by the nucleotide sequence shownin SEQ ID NO: 233 was calculated to be 45830 Da. The molecular weight ofthe protein consisting of the amino acid sequence (SEQ ID NO: 253)encoded by te nucleotide sequence shown in SEQ ID NO: 263 was calculatedto be 7034 Da.

Example 65 Expression of the Present Invention DNA (A24) in E. Coli

(1) Production of a Transformed E. coli Having the Present Invention DNA(A24)

PCR was conducted similarly to Example 49(1), other than utilizing as atemplate the chromosomal DNA prepared from Streptomyces rutgersensis IFO15875T in Example 64(1) and utilizing as the printers theoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 320and an oligonucleotide having the nucleotide sequence shown in SEQ IDNO: 321. Similarly to Example 32(1), the DNA was purified from thereaction solution of PCR and cloned into the cloning vector pCRII-TOPO(Invitrogen Company). The nucleotide sequence of the obtained plasmidDNA was sequenced by utilizing as primers the oligonucleotides havingthe nucleotide sequences shown, respectively, in SEQ ID NOs: 67, 68 and322. Based on the obtained results, the plasmid having the nucleotidesequence shown in SEQ ID NO: 243 was designated as pCR1589BF. Similarlyto Example 32(1), pCR1589BF was digested with restriction enzymes NdeIand HindIII. A DNA of about 1.5 kbp was purified from the digestionproducts. The obtained DNA and the plasmid pKSN2 digested with NdeI andHindIII were ligated to obtain a plasmid containing the nucleotidesequence shown in SEQ ID NO: 243, in which the DNA encoding the presentinvention protein (A24) is inserted between the NdeI site and theHindIII site of pKSN2 (hereinafter referred to as “pKSN1589BF”). Saidplasmid was introduced into E. Coli JM109. The obtained E. colitransformant was designated JM109/pKSN1589BF.

(2) Expression of the Present Invention Protein (A24) in E. coli andRecovery of Said Protein

Similarly to Example 4(2), each of E. coli JM109/pKSN1589BF andJM109/pKSN2 was cultured. The cells were recovered. Cell lysatesolutions were prepared. Under the method described in Example 4(2),supernatant fractions were prepared from the cell lysate solutions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN1589SF is referred to as “E. coli pKSN1589BF extract” and thesupernatant fraction obtained from E. coli JM109/pKSN2 is referred to as“E. coli pKSN2 extract”).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Similarly to Example 32(3), reaction solutions of 30 μl were preparedand maintained for 10 minutes at 30° C. However, as the supernatantfraction, the supernatant fraction prepared in Example 65(2) (E. colipKSN1589BF extract or E. coli pKSN2 extract) was utilized. The reactionsolutions after the maintenance were extracted with ethyl acetate andthe extracted layers were TLC analyzed. After developing the TLC plate,the presence of a spot thereon corresponding to compound (III) labeledwith ¹⁴C were examined (Rf value 0.24 and 0.29). A spot corresponding tocompound (III) was detected from the reaction solution containing E.coli pKSN1589BF extract. In contrast, such a spot was not detected fromthe reaction solution containing E. coli pKSN2 extract.

Example 66 Obtaining the Present Invention DNA (A25)

(1) Preparation of the Chromosomal DNA of Streptomyces steffisburgensisIFO 13446T

Under the method described in Example 31(1), the chromosomal DNA ofStreptomyces steffisburgensis IFO 13446T was prepared.

(2) Isolation of DNA Having a Partial Nucleotide Sequence of the PresentInvention DNA (A25)

PCR was conducted by utilizing as the template the chromosomal DNA ofStreptomyces steffisburgensis IFO 13446T prepared in Example 66(1) andby utilizing primer pairing 14, in accordance with the method describedin Example 29. Similarly to Example 31(2), the amplified DNA was clonedto cloning vector pCRII-TOPO (Invitrogen Company). The nucleotidesequence thereof was analyzed. As a result, the nucleotide sequenceshown in nucleotides 289 to 1015 of the nucleotide sequence shown in SEQID NO: 234 was provided.

Further, the chromosomal DNA prepared in Example 66(1) was digested withrestriction enzyme SmaI. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 323 and primer AP1. Next PCR was conductedunder the conditions described in Example 26(3), by utilizing the firsPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 324 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 1 to 303 of the nucleotide sequence shownin SEQ ID NO: 244 was provided.

Further, the chromosomal DNA prepared in Example 66(1) was digested withrestriction enzyme PmacI. A genome walker library was produced byutilizing the obtained DNA, according to the method described in Example26(3). PCR was conducted under the conditions described in Example 26(3)to obtain the first PCR products, by utilizing the obtained library asthe template and by utilizing the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 311 and primer AP1. Next, PCR was conductedunder the conditions described in Example 26(3), by utilizing the firstPCR products as the template and by utilizing the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 325 and primer AP2. Thenucleotide sequence of the obtained DNA was analyzed. The nucleotidesequence shown in nucleotides 966 to 1411 of the nucleotide sequenceshown in SEQ ID NO: 244 was provided.

(3) Sequence Analysis of the Present Invention DNA (A25)

The nucleotide sequence shown in SEQ ID NO: 244 was obtained byconnecting the nucleotide sequences provided by the DNA obtained inExample 66(2). Two open reading frames (ORF) were present in saidnucleotide sequence. As such, there was contained a nucleotide sequence(SEQ ID NO: 234) consisting of 1197 nucleotides (inclusive of the stopcodon) and encoding a 398 amino acid residue (SEQ ID NO: 224) and anucleotide sequence (SEQ ID NO: 264) consisting of 201 nucleotides(inclusive of the stop codon) and encoding a 66 amino acid residue (SEQID NO: 254). The molecular weight of the protein consisting of the aminoacid sequence (SEQ ID NO: 224) encoded by the nucleotide sequence shownin SEQ ID NO: 234 was calculated to be 44175 Da. The molecular weight ofthe protein consisting of the amino acid sequence (SEQ ID NO: 254)encoded by the nucleotide sequence shown in SEQ ID NO: 264 wascalculated to be 6685 Da.

Example 67 Expression of the Present Invention DNA (A25) in E. Coli

(1) Production of a Transformed E. coli Having the Present Invention DNA(A25)

PCR was conducted similarly to Example 49(1), other than utilizing as atemplate the chromosomal DNA prepared from Streptomyces steffisburgensisIFO 13446T in Example 66(1) and utilizing as the primers theoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 326and an oligonucleotide having the nucleotide sequence shown in SEQ IDNO: 327 Similarly to Example 32(1), the DNA was purified from thereaction solution of PCR and cloned into the cloning vector pCRII-TOPO(Invitrogen Company). The nucleotide sequence of the obtained plasmidDNA was sequenced by utilizing as primers the oligonucleotides havingthe nucleotide sequences shown, respectively, in SEQ ID NOs: 67, 68,311, 315 and 323, Based on the obtained results, the plasmid having thenucleotide sequence shown in SEQ ID NO; 244 was designated as pCR1609F.Similarly to Example 32(1), pCR1609F was digested with restrictionenzymes NdeI and HindIII. A DNA of about 1.5 kbp was purified from thedigestion products. The obtained DNA and the plasmid pKSN2 digested withNdeI and HindIII were ligated to obtain a plasmid containing thenucleotide sequence shown in SEQ ID NO: 244, in which the DNA encodingthe present invention protein (A25) is inserted between the NdeI siteand the HindIII site of pKSN2 (hereinafter referred to as “pKSN1609F”).Said plasmid was introduced into E. Coli JM109. The obtained E. colitransformant was designated JM109/pKSN1609F.

(2) Expression of the Present Invention Protein (A25) in E. coli andRecovery of Said Protein

Similarly to Example 4(2), each of E. coli JM109/pKSN1609F andJM109/pKSN2 was cultured. The cells were recovered. Cell lysatesolutions were prepared. Under the method described in Example 4(2),supernatant fractions were prepared from the cell lysate solutions(hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN1609F is referred to as “E. coli pKSN1609F extract” and thesupernatant fraction obtained from E. coli JM109/pKSN2 is referred to as“E. coli pKSN2 extract”).

(3) Detection of the Ability to Convert Compound (II) to Compound (III)

Similarly to Example 32(3), reaction solutions of 30 μl were preparedand maintained for 10 minutes at 30° C. However, as the supernatantfraction, the supernatant fraction prepared in Example 67(2) (E. colipKSN1609F extract or E. coli pKSN2 extract) was utilized. The reactionsolutions after the maintenance were extracted with ethyl acetate andthe extracted layers were TLC analyzed. After developing the TLC plate,the presence of a spot thereon corresponding to compound (III) labeledwith ¹⁴C were examined (Rf value 0.24 and 0.29). A spot corresponding tocompound (III) was detected from the reaction solution containing E.coli pKSN1609F extract. In contrast, such a spot was not detected fromthe reaction solution containing E. coli pKSN2 extract.

Example 68 Metabolism of Compounds by the Present Invention Protein(A16), (A17), (A18), (A19), (A20), (A21), (A22), (A23), (A24) or (A25)

(1) Metabolism of Compound (XI) by the Present Invention Protein (A16)

There was prepared 100 μl of a reaction solution of 50 mM potassiumphosphate buffer (pH7.0) containing 12.5 ppm of compound (XII), 3 mM ofa β-NADPH (hereinafter, referred to as “component A”) (Oriental YeastCompany), 1 mg/ml of a ferredoxin derived from spinach (hereinafterreferred to as “component B”) (Sigma Company), 0.15 U/ml of ferredoxinreductase (hereinafter, referred to as “component C”) (Sigma Company)and 20 μl of the supernatant fraction recovered in Example 49(2). Thereaction solution was maintained at 30° C. for 10 minutes. Further,there was prepared and maintained similarly 100 μl of a reactionsolution of a 50 mM potassium phosphate buffer (pH 7.0) having noaddition of at least one component utilized in the composition of theabove reaction solution, selected from component A, component 13,component C and the supernatant fraction prepared in Example 49(2). Fivemicroliters (5 μl) of 2N HCl and 100 μl of ethyl acetate were added andmixed into each of the reaction solutions after the maintenance. Thesupernatant centrifuged at 8,000×g was filtered with UltraFree MC 0.22μm filter unit (Millipore Company) Forty microliters (40 μl) of theliquid filtrate (hereinafter, the liquid filtrate derived from thereaction solution containing component A, component B, component C and20 μl of supernatant fraction recovered in Example 49(2) is referred toas “(XII) metabolism solution (A16)”; further, the liquid filtratederived from the reaction solution containing no component A, nocomponent B, no component C and no supernatant fraction recovered inExample 49(2) is referred to as “(XII) control solution (A16)”) wereanalyzed on a HPLC under the above analysis condition 1. Compared to theconcentration of compound (XII) detected from (XII) control solution(A16), the concentration of compound (XII) detected from (XII)metabolism solution (A16) was lower. Further a peak, which was notdetected from the (XII) control solution (A16), was detected from the(XII) metabolism solution (A16). The elution time of said peak on theHPLC matched an elution time of a peak of a compound in which the massis 14 less than said compound (XII) detected from (XII) metabolismsolution (A1) in Example 41(2).

(2) Metabolism of Compound (XII) by the Present Invention Protein (A17)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 51(2) instead of 20 μl of the supernatant fraction recovered inExample 49(2), the reaction solution was prepared and maintained inaccordance with the method described in Example 68(1). Similar toExample 68(1), the reaction solution after the maintenance was prepared.Forty microliters (40 μl) of the obtained liquid filtrate (hereinafter,the liquid filtrate derived from the reaction solution containingcomponent A, component B, component C and 20 μl of supernatant fractionrecovered in Example 51(2) is referred to as “(XII) metabolism solution(A17)”; further, the liquid filtrate derived from the reaction solutioncontaining no component A, no component B, no component C and nosupernatant fraction recovered in Example 51(2) is referred to as “(XII)control solution (A17)”) were analyzed on a HPLC under the aboveanalysis condition 1. Compared to the concentration of compound (XII)detected from (XII) control solution (A17), the concentration ofcompound (XII) detected from (XII) metabolism solution (A17) was lower.Further a peak, which was not detected from the (XII) control solution(A17), was detected from the (XII) metabolism solution (A17). Theelution time of the said peak on the HPLC matched an elution time of apeak of a compound in which the mass is 14 less than said compound (XII)detected from (XII) metabolism solution (A1) in Example 41(2).

(3) Metabolism of Compound (XII) by the Present Invention Protein (A18)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 53(2) instead of 20 μl of the supernatant fraction recovered inExample 49(2), the reaction solution was prepared and maintained inaccordance with the method described in Example 68(1). Similar toExample 68(1), each of the reaction solutions after the maintenance wasprepared. Forty microliters (40 μl) of the obtained liquid filtratehereinafter, the liquid filtrate derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 53(2) is referred to as “(XII)metabolism solution (A18)”; further, the liquid filtrate derived fromthe reaction solution containing no component A, no component 13, nocomponent C and no supernatant fraction recovered in Example 53(2) isreferred to as “(XII) control solution (A18)”) was analyzed on a HPLCunder analysis condition 1. Compared to the concentration of compound(XII) detected from (XII) control solution (A18), the concentration ofcompound (XII) detected from (XII) metabolism solution (A18) was lower.Further a peak, which was not detected from the (XII) control solution(A18), was detected from the (XII) metabolism solution (A18). Theelution time of said peak on the HPLC matched an elution time of a peakof a compound in which the mass is 14 less than said compound (XII)detected from (XII) metabolism solution (A1) in Example 41(2).

(4) Metabolism of Compound (XII) by the Present Invention Protein (A19)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 55(2) instead of 20 μl of the supernatant fraction recovered inExample 49(2), the reaction solution was prepared and maintained inaccordance with the method described in Example 68(1). Similar toExample 68(1), each of the reaction solutions after the maintenance wasprepared. Forty microliters (40 μl) of the obtained liquid filtrate(hereinafter, the liquid filtrate derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 55(2) is referred to as “(XII)metabolism solution (A19)”; further, the liquid filtrate derived fromthe reaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 55(2) isreferred to as “(XII) control solution (A19)”) was analyzed on a HPLCunder analysis condition 1. Compared to the concentration of compound(XII) detected from (XII) control solution (A19), the concentration ofcompound (XII) detected from (XII) metabolism solution (A19) was lower.Further a peak, which was not detected from the (XII) control solution(A19), was detected from the (XII) metabolism solution (A19). Theelution time of said peak on the HPLC matched an elution time of a peakof a compound in which the mass is 14 less than said compound (XII)detected from (XII) metabolism solution (A1) in Example 41(2).

(5) Metabolism of Compound (XIII) by the Present Invention Protein (A20)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 57(2) instead of 20 μl of the supernatant fraction recovered inExample 49(2), the reaction solution was prepared and maintained inaccordance with the method described in Example 68(1). Similar toExample 68(1), each of the reaction solutions after the maintenance wasprepared. Forty microliters (40 μl) of the obtained liquid filtrate(hereinafter, the liquid filtrate derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 57(2) is referred to as “(XII)metabolism solution (A20)”; further, the liquid filtrate derived fromthe reaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 57(2) isreferred to as “(XII) control solution (A20)”) was analyzed on a HPLCunder analysis condition 1. Compared to the concentration of compound(XII) detected from (XII) control solution (A20), the concentration ofcompound (XII) detected from (XII) metabolism solution (A20) was lower.Further a peak, which was not detected from the (XII) control solution(A20), was detected from the (XII) metabolism solution (A20) The elutiontime of said peak on the HPLC matched an elution time of a peak of acompound in which the mass is 14 less than said compound (XII) detectedfrom (XII) metabolism solution (A1) in Example 41(2).

(6) Metabolism of Compound (XII) by the Present Invention Protein (A21)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 59(2) instead of 20 μl of the supernatant fraction recovered inExample 49(2), the reaction solution was prepared and maintained inaccordance with the method described in Example 68(1). Similar toExample 68(1), each of the reaction solutions after the maintenance wasprepared. Forty microliters (40 μl) of the obtained liquid filtrate(hereinafter, the liquid filtrate derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 59(2) is referred to as “(XII)metabolism solution (A21)”; further, the liquid filtrate derived fromthe reaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 59(2) isreferred to as “(XII) control solution (A21)”) was analyzed on a HPLCunder analysis condition 1. Compared to the concentration of compound(XII) detected from (XII) control solution (A21), the concentration ofcompound (XII) detected from (XII) metabolism solution (A21) was lower.Further a peak, which was not detected from the (XII) control solution(A21), was detected from the (XII) metabolism solution (A21). Theelution time of said peak on the HPLC matched an elution time of a peakof a compound in which the mass is 14 less than said compound (XII)detected from (XII) metabolism solution (A1) in Example 41(2).

(7) Metabolism of Compound (XII) by the Present Invention Protein (A22)

Other than utilizing 201 μl of the supernatant fraction recovered inExample 61(2) instead of 20 μl of the supernatant fraction recovered inExample 49(2), the reaction solution was prepared and maintained inaccordance with the method described in Example 68(1). Similar toExample 68(1), each of the reaction solutions after the maintenance wasprepared. Forty microliters (40 μl) of the obtained liquid filtrate(hereinafter, the liquid filtrate derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 61(2) is refereed to as “(XII)metabolism solution (A22)”; further, the liquid filtrate derived fromthe reaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 61(2) isreferred to as “(XII) control solution (A22)”) was analyzed on a HPLCunder analysis condition 1. Compared to the concentration of compound(XII) detected from (XII) control solution (A22), the concentration ofcompound (XII) detected from (XII) metabolism solution (A22) was lower.Further a peak, which was not detected from the (XII) control solution(A22), was detected from the (XII) metabolism solution (A22). Theelution time of said peak on the HPLC matched an elution time of a peakof a compound in which the mass is 14 less than said compound (XII)detected from (XII) metabolism solution (A1) in Example 41(2).

(8) Metabolism of Compound (XII) by the Present Invention Protein (A23)

Other than utilizing 20 μl of the Supernatant Fraction Recovered inExample 63(2) instead of 20 μl of the supernatant fraction recovered inExample 49(2), the reaction solution was prepared and maintained inaccordance with the method described in Example 68(1). Similar toExample 68(1), each of the reaction solutions after the maintenance wasprepared. Forty microliters (40 μl) of the obtained liquid filtrate(hereinafter, the liquid filtrate derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 63(2) is referred to as “(XII)metabolism solution (A23)”; further, the liquid filtrate derived fromthe reaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 63(2) isreferred to as “(XII) control solution (A23)”) was analyzed on a HPLCunder analysis condition 1. Compared to the concentration of compound(XII) detected from (XII) control solution (A23), the concentration ofcompound (XI) detected from (XII) metabolism solution (A23) was lower.Further a peak, which was not detected from the (XII) control solution(A23), was detected from the (XII) metabolism solution (A23). Theelution time of said peak on the HPLC matched an elution time of a peakof a compound in which the mass is 14 less than said compound (XII)detected from (XII) metabolism solution (A1) in Example 41(2).

(9) Metabolism of Compound (XII) by the Present Invention Protein (A24)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 65(2) instead of 20 μl of the supernatant fraction recovered inExample 49(2), the reaction solution was prepared and maintained inaccordance with the method described in Example 68(1). Similar toExample 68(1), each of the reaction solutions after the maintenance wasprepared. Forty microliters (40 μl) of the obtained liquid filtrate(hereinafter, the liquid filtrate derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 65(2) is referred to as “(XII)metabolism solution (A24)”; further, the liquid filtrate derived fromthe reaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 65(2) isreferred to as “(XII) control solution (A24)”) was analyzed on a HPLCunder analysis condition 1. Compared to the concentration of compound(XII) detected from (XII) control solution (A24), the concentration ofcompound (XII) detected from (XII) metabolism solution (A24) was lower.Further a peak, which was not detected from the (XII) control solution(A24), was detected from the (XII) metabolism solution (A24). Theelution time of said peak on the HPLC matched an elution time of a peakof a compound in which the mass is 14 less than said compound (XII)detected from (XII) metabolism solution (A1) in Example 41(2).

(10) Metabolism of Compound (XII) by the Present Invention Protein (A25)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 67(2) instead of 20 μl of the supernatant fraction recovered inExample 49(2), the reaction solution was prepared and maintained inaccordance with the method described in Example 68(1). Similar toExample 68(1), each of the reaction solutions after the maintenance wasprepared. Forty microliters (40 μl) of the obtained liquid filtrate(hereinafter, the liquid filtrate derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 67(2) is referred to as “(XII)metabolism solution (A25)”; further, the liquid filtrate derived fromthe reaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 67(2) isreferred to as “(XII) control solution (A25)”) was analyzed on a HPLCunder analysis condition 1. Compared to the concentration of compound(XII) detected from (XII) control solution (A25), the concentration ofcompound (XII) detected from (XII) metabolism solution (A25) was lower.Further a peak, which was not detected from the (XII) control solution(A25), was detected from the (XII) metabolism solution (A25). Theelution time of said peak on the HPLC matched an elution time of a peakof a compound in which the mass is 14 less than said compound (XII)detected from (XIX) metabolism solution (A1) in Example 41(2).

(11) Metabolism of Compound (XIII) by the Present Invention Protein(A17)

Other than utilizing 12.5 ppm of compound (XIII) instead of 12.5 ppm ofcompound (XII), the reaction solution was prepared and maintained inaccordance with the method described in Example 68(2). Similar toExample 68(1), each of the reaction solutions after the maintenance wasprepared. Forty microliters (40 μl) of the obtained liquid filtrate(hereinafter, the liquid filtrate derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 51(2) is referred to as“(XIII) metabolism solution (A17)”; further, the liquid filtrate derivedfrom the reaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 51(2) isreferred to as “(XIII) control solution (A17)”) were analyzed on a HPLCunder the above analysis condition 1. Compared to the concentration ofcompound (XIII) detected from (XIII) control solution (A17), theconcentration of compound (XIII) detected form (XIII) metabolismsolution (A17) was lower. Further a peak, which was not detected fromthe (XIII) control solution (A17), was detected from the (XIII)metabolism solution (A17). The elution time of the said peak on the HPLCmatched an elution time of a peak of a compound in which the mass is 14less than said compound (XIII) detected from (XIII) metabolism solution(A1) in Example 41(3),

(12) Metabolism of Compound (XIII) by the Present Invention Protein(A18)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 53(2) instead of 20 μl of the supernatant fraction recovered inExample 51(2), the reaction solution was prepared and maintained inaccordance with the method described in Example 68(11). Similar toExample 68(1), each of the reaction solutions after the maintenance wasprepared. Forty microliters (40 μl) of the obtained liquid filtrate(hereinafter, the liquid filtrate derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 53(2) is referred to as“(XIII) metabolism solution (A18)”; further, the liquid filtrate derivedfrom the reaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 53(2) isreferred to as “(XIII) control solution (A18)”) was analyzed on a HPLCunder analysis condition 1. Compared to the concentration of compound(XIII) detected from (XIII) control solution (A18), the concentration ofcompound (XIII) detected from (XIII) metabolism solution (A18) waslower. Further a peak, which was not detected from the (XIII) controlsolution (A18), was detected from the (XIII) metabolism solution (A18).The elution time of said peak on the HPLC matched an elution time of apeak of a compound in which the mass is 14 less than said compound(XIII) detected from (XIII) metabolism solution (A1) in Example 41(3).

(13) Metabolism of Compound (XIII) by the Present Invention Protein(A19)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 55(2) instead of 20 μl of the supernatant fraction recovered inExample 51(2), the reaction solution was prepared and maintained inaccordance with the method described in Example 68(11). Similar toExample 68(1), each of the reaction solutions after the maintenance wasprepared. Forty microliters (40 μl) of the obtained liquid filtrate(hereinafter, the liquid filtrate derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 55(2) is referred to as“(XIII) metabolism solution (A19)”; further, the liquid filtrate derivedfrom the reaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 55(2) isreferred to as “(XIII) control solution (A19)”) was analyzed on a HPLCunder analysis condition 1. Compared to the concentration of compound(XIII) detected from (XIII) control solution (A19), the concentration ofcompound (XIII) detected from (XIII) metabolism solution (A19) waslower. Further a peak, which was not detected from the (XIII) controlsolution (A19), was detected from the (XII) metabolism solution (A19).The elution time of said peak on the HPLC matched an elation time of apeak of a compound in which the mass is 14 less than said compound(XIII) detected from (XIII) metabolism solution (A1) in Example 41(3).

(14) Metabolism of Compound (XIII) by the Present Invention Protein(A20)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 57(2) instead of 20 μl of the supernatant fraction recovered inExample 51(2), the reaction solution was prepared and maintained inaccordance with the method described in Example 68(11). Similar toExample 68(1), each of the reaction solutions after the maintenance wasprepared. Forty microliters (40 μl) of the obtained liquid filtrate(hereinafter, the liquid filtrate derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 57(2) is referred to as“(XIII) metabolism solution (A20)”; further, the liquid filtrate derivedfrom the reaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 57(2) isreferred to as “(XIII) control solution (A20)”) was analyzed on a HPLCunder analysis condition 1. Compared to the concentration of compound(XIII) detected from (XIII) control solution (A20), the concentration ofcompound (XIII) detected from (XIII) metabolism solution (A20) waslower. Further a peak, which was not detected from the (XIII) controlsolution (A20), was detected from the (XIII) metabolism solution (A20).The elution time of said peak on the HPLC matched an elution time of apeak of a compound in which the mass is 14 less than said compound(XIII) detected from (XI) metabolism solution (A1) in Example 41(3).

(15) Metabolism of Compound (XIII) by the Present Invention Protein(A21)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 59(2) instead of 20 μl of the supernatant fraction recovered inExample 51(2), the reaction solution was prepared and maintained inaccordance with the method described in Example 68(11). Similar toExample 68(1), each of the reaction solutions after the maintenance wasprepared. Forty microliters (40 μI) of the obtained liquid filtrate(hereinafter, the liquid filtrate derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 59(2) is referred to as“(XIII) metabolism solution (A21)”; further, the liquid filtrate derivedfrom the reaction solution containing no component A, no component 13,no component C and no supernatant fraction recovered in Example 59(2) isreferred to as “(XIII) control solution (A21)”) was analyzed on a HPLCunder analysis condition 1. Compared to the concentration of compound(XIII) detected from (XIII) control solution (A21), the concentration ofcompound (XIII) detected from (XIII) metabolism solution (A21) waslower. Further a peak, which was not detected from the (XIII) controlsolution (A21), was detected from the (XIII) metabolism solution (A21).The elution time of said peak on the HPLC matched an elution time of apeak of a compound in which the mass is 14 less than said compound(XIII) detected from (XIII) metabolism solution (A1) in Example 41(3).

(16) Metabolism of Compound (XIII) by the Present Invention Protein(A23)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 63(2) instead of 20 μl of the supernatant fraction recovered inExample 51(2), the reaction solution was prepared and maintained inaccordance with the method described in Example 68(11). Similar toExample 68(1), each of the reaction solutions after the maintenance wasprepared. Forty microliters (40 μl) of the obtained liquid filtrate(hereinafter, the liquid filtrate derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 63(2) is referred to as“(XIII) metabolism solution (A23)”; further, the liquid filtrate derivedfrom the reaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 63(2) isreferred to as “(XIII) control solution (A23)”) was analyzed on a HPLCunder analysis condition 1. Compared to the concentration of compound(XIII) detected from (XIII) control solution (A23), the concentration ofcompound (XIII) detected from (XIII) metabolism solution (A23) was lowerFurther a peak, which was not detected from the (XIII) control solution(A23), was detected from the (XIII) metabolism solution (A23). Theelution time of said peak on the HPLC matched an elution time of a peakof a compound in which the mass is 14 less than said compound (XIII)detected from (XIII) metabolism solution (A1) in Example 41(3).

(17) Metabolism of Compound (XIII) by the Present Invention Protein(A25)

Other than utilizing 20 μl of the supernatant fraction recovered inExample 67(2) instead of 20 μl of the supernatant fraction recovered inExample 51(2), the reaction solution was prepared and maintained inaccordance with the method described in Example 68(11). Similar toExample 68(1), each of the reaction solutions after the maintenance wasprepared. Forty microliters (40 μl) of the obtained liquid filtrate(hereinafter, the liquid filtrate derived from the reaction solutioncontaining component A, component B, component C and 20 μl ofsupernatant fraction recovered in Example 67(2) is referred to as “(XII)metabolism solution (A25)”; further, the liquid filtrate derived fromthe reaction solution containing no component A, no component B, nocomponent C and no supernatant fraction recovered in Example 67(2) isreferred to as “(XIII) control solution (A25)”) was analyzed on a HPLCunder analysis condition 1. Compared to the concentration of compound(XIII) detected from (XIII) control solution (A25), the concentration ofcompound (XIII) detected from (XIII) metabolism solution (A25) waslower. Further a peak, which was not detected from the (XIII) controlsolution (A25), was detected from the (XIII) metabolism solution (A25).The elution time of said peak on the HPLC matched an elution time of apeak of a compound in which the mass is 14 less than said compound(XIII) detected from (XIII) metabolism solution (A1) in Example 41(3).

Example 69 Hybridization in Which the Present Invention DNA (A1), (A2),(A3) or (A4) was a Probe

(1) Preparation of a Probe

PCR was conducted in accordance with the method described in Example30(1). However, as the template, 10 ng of the chromosomal DNA ofStreptomyces achromogenes IFO 12735 prepared in Example 26(1) wasutilized instead of said 50 ng of the chromosomal DNA of Streptomycesphaeochromogenes IFO12898 prepared in Example 3(1). As the primers,there was utilized an oligonucleotide having the nucleotide sequenceshown in SEQ ID NO: 328 and the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 329. The DNA amplified by said PCR wasrecovered to produce a probe having the nucleotide sequence shown in SEQID NO: 109 labeled with digoxigenin (hereinafter referred to as “DIGlabeled probe (A4)”).

(2) Preparation of the Plasmid Solution

PCR was conducted by utilizing Advantage-GC genomic polymerase mix(Clontech Company) and by utilizing as the template the chromosomal DNAof Streptomyces nogalator IFO13445 prepared in Example 31(1). As theprimers, there was utilized the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 330 and the oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 331. The PCR reaction solutionamounted to 50 μl by adding the 2 primers each amounting to 200 nM, 10ng of the chromosomal DNA, 4.0 μl of dNTP mix (a mixture of 2.5 mm ofeach of the 4 types of dNTP; Clontech Company), 1.0 μl of 5×GC buffer,2.2 μl of 25 mM Mg(OAc)₂, 10.0 μl of 5M GC-Melt and 1.0 μl ofAdvantage-GC genomic polymerase mix (Clontech Company) and distilledwater. The reaction conditions of the PCR were after maintaining 94° C.for 1 minute; repeating 7 cycles of a cycle that included maintaining94° C. for 10 seconds and then 72° C. for 3 minutes; repeating 36 cyclesof a cycle that included 94° C. for 10 seconds and then 67° C. for 3minutes; and then maintaining 67° C. for 7 minutes. The DNA was purifiedfrom the PCR reaction solution with QIAquick PCR Purification Kit(Qiagen Company) according to the instructions attached to said kit. Theobtained DNA was ligated to TA cloning vector pCR2.1 (InvitrogenCompany), according to the attached manual, and was introduced into E.Coli TOP10F′ (Invitrogen Company). The plasmid DNA was prepared from theobtained E. coli transformant, utilizing QIAprep Spin Miniprep Kit(Qiagen Company) to obtain a plasmid solution containing the presentinvention DNA (A11).

Similarly, PCR was conducted by utilizing as the template thechromosomal DNA of Streptomyces tsusimaensis IFO 13782 prepared inExample 33(1) and by utilizing as primers the oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 332 and the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 333. The DNA obtainedby the PCR was ligated to the vector similar to the above. E. coli wasthen transformed. The plasmid was prepared from the obtained E. colitransformant to obtain a plasmid solution containing the presentinvention DNA (A12).

Similarly, PCR was conducted by utilizing as the template thechromosomal DNA of Streptomyces thermocoerulesces IFO 14273t prepared inExample 35(1) and by utilizing as primers the oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 331 and the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 334. The DNA obtainedby the PCR was ligated to the vector similar to the above. E. coli wasthen transformed. The plasmid was prepared from the obtained E. colitransformant to obtain a plasmid solution containing the presentinvention DNA (A13).

Similarly, PCR was conducted by utilizing as the template thechromosomal DNA of Streptomyces glomerochromogenes IFO 13673t preparedin Example 37(1) and by utilizing as primers the oligonucleotide havingthe nucleotide sequence shown in SEQ AD NO: 330 and the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 331. The DNA obtainedby the PCR was ligated to the vector similar to the above. E. coli wasthen transformed. The plasmid was prepared from the obtained E. colitransformant to obtain a plasmid solution containing the presentinvention DNA (A14).

Similarly, PCR was conducted by utilizing as the template thechromosomal DNA of Streptomyces olivochromogenes IFO 12444 prepared inExample 39(1) and by utilizing as primers the oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 330 and the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 331. The DNA obtainedby the PCR was ligated to the vector similar to the above. E. coli wasthen transformed. The plasmid was prepared from the obtained E. colitransformant to obtain a plasmid solution containing the presentinvention DNA (A15).

Similarly, PCR was conducted by utilizing as the template thechromosomal DNA of Streptomyces ornatus IFO 13069′ prepared in Example48(1) and by utilizing as primers the oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 335 and the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 336. The DNA obtainedby the PCR was ligated to the vector similar to the above. E. coli wasthen transformed. The plasmid was prepared from the obtained E. colitransformant to obtain a plasmid solution containing the presentinvention DNA (A16).

Similarly, PCR was conducted by utilizing as the template thechromosomal DNA of Streptomyces griseus ATCC 10137 prepared in Example50(1) and by utilizing as primers the oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 335 and the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 336. The DNA obtainedby the PCR was ligated to the vector similar to the above. E. coli wasthen transformed. The plasmid was prepared from the obtained E. colitransformant to obtain a plasmid solution containing the presentinvention DNA (A 17).

Similarly, PCR was conducted by utilizing as the template thechromosomal DNA of Streptomyces achromogenes IFO 12735 prepared inExample 52(1) and by utilizing as primers the oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 330 and the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 331. The DNA obtainedby the PCR was ligated to the vector similar to the above. E. coli wasthen transformed. The plasmid was prepared from the obtained E. colitransformant to obtain a plasmid solution containing the presentinvention DNA (A 18).

Similarly, PCR was conducted by utilizing as the template thechromosomal DNA of Streptomyces griseus IFO 13849T prepared in Example54(1) and by utilizing as primers the oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 333 and the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 335. The DNA obtainedby the PCR was ligated to the vector similar to the above E. coli wasthen transformed. The plasmid was prepared from the obtained E. colitransformant to obtain a plasmid solution containing the presentinvention DNA (A19).

Similarly, PCR was conducted by utilizing as the template thechromosomal DNA of Streptomyces lanatus IFO 12787T prepared in Example56(1) and by utilizing as primers the oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 331 and the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 337. The DNA obtainedby the PCR was ligated to the vector similar to the above. E. coli wasthen transformed. The plasmid was prepared from the obtained E. colitransformant to obtain a plasmid solution containing the presentinvention DNA (A20).

Similarly, PCR was conducted by utilizing as the template thechromosomal DNA of Streptomyces misawanensis IFO 13855T prepared inExample 58(1) and by utilizing as primers the oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 331 and the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 338. The DNA obtainedby the PCR was ligated to the vector similar to the above. E. coli wasthen transformed. The plasmid was prepared from the obtained E. colitransformant to obtain a plasmid solution containing the presentinvention DNA (A21).

Similarly, PCR was conducted by utilizing as the template thechromosomal DNA of Streptomyces roseorubens IFO 13682T prepared inExample 62(1) and by utilizing as primers the oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 331 and the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 339. The DNA obtainedby the PCR was ligated to the vector similar to the above. E. coli wasthen transformed. The plasmid was prepared from the obtained E. colitransformant to obtain a plasmid solution containing the presentinvention DNA (A23).

Similarly, PCR was conducted by utilizing as the template thechromosomal DNA of Streptomyces steffisburgensis IFO 13446T prepared inExample 66(1) and by utilizing as primers the oligonucleotide having thenucleotide sequence shown in SEQ JD NO: 331 and the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 339. The DNA obtainedby the PCR was ligated to the vector similar to the above. E. coli wasthen transformed. The plasmid was prepared from the obtained E. colitransformant to obtain a plasmid solution containing the presentinvention DNA (A25).

Further, similarly, PCR was conducted by utilizing as the template thechromosomal DNA of Streptomyces pallidus IFO 13434T prepared in Example60(1) and by utilizing as primers the oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 340 and the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 341. The DNA obtainedby the PCR was ligated to the vector similar to the above. E. coli wasthen transformed. The plasmid was prepared from the obtained E. colitransformant to obtain a plasmid solution containing the presentinvention DNA (A22).

Similarly, PCR was conducted by utilizing as the template thechromosomal DNA of Streptomyces rutgersensis IFO 15875T prepared inExample 64(1) and by utilizing as primers the oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 342 and the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 343. The DNA obtainedby the PCR was ligated to the vector similar to the above. E. coli wasthen transformed. The plasmid was prepared from the obtained E. colitransformant to obtain a plasmid solution containing the presentinvention DNA (A24).

(2) Dot Blot Hybridization

About 100 ng and 10 ng of each of the plasmids prepared in Example 69(2)was blotted on a Hybond N+ Nylon Membrane (Amersham BiosciencesCompany). The plasmids were: the plasmid DNA containing the presentinvention DNA (A11), the plasmid DNA containing the present inventionDNA (A12), the plasmid DNA containing the present invention DNA (A13),the plasmid DNA containing the present invention DNA (A14), the plasmidDNA containing the present invention DNA (A15), the plasmid DNAcontaining the present invention DNA (A16), the plasmid DNA containingthe present invention DNA (A17), the plasmid DNA containing the presentinvention DNA (A18), the plasmid DNA containing the present inventionDNA (A19), the plasmid DNA containing the present invention DNA (A20),the plasmid DNA containing the present invention DNA (A21), the plasmidDNA containing the present invention DNA (A23), and the plasmid DNAcontaining the present invention DNA (A25). Ultraviolet light wasdirected at the obtained membranes with a transilluminator for 5minutes.

Hybridization and detection were conducted according to the methoddescribed in Example 30(2). The probes prepared in Example 30(1) weremaintained at 100° C. for 5 minutes and then cooled on ice. As theprobes, there was utilized the DNA having the nucleotide sequence shownin SEQ ID NO: 6 labeled with digoxigenin (hereinafter referred to as“DIG labeled probe (A1)”), the DNA having the nucleotide sequence shownin SEQ ID NO: 7 labeled with digoxigenin (hereinafter referred to as“DIG labeled probe (A2)”), the DNA having the nucleotide sequence shownin SEQ ID NO: 8 labeled with digoxigenin (hereinafter referred to as“DIG labeled probe (A3)”) or the DIG labeled probe (A4) produced inExample 69(1). In the events of utilizing any one of the DIG labeledprobe (A1), (A2), (A3) or (A4) for hybridization, a signal was detectedfor each of the reagents of the 10 ng and 100 ng of each of the aboveplasmid DNA.

Further, similarly, about 10 ng and 100 ng of each of the plasmid DNAcontaining the present invention DNA (A22) prepared in Example 69(2) andthe plasmid DNA containing the present invention DNA (A24) are blottedonto a Hybond N+ nylon membrane (Amersham Biosciences Company).Hybridization and detection are conducted accordingly to Example 30(2).

Example 70 Preparation of the Present Invention DNA (A23) in Which theCodon Usage has been Adjusted for Expression in Soybean (Hereinafter,Referred to as the “Present Invention DNA (A23)S”)

(1) Preparation of the Present Invention DNA (A23)S

PCR was conducted with Pyrobest DNA polymerase (Takara Shuzo Company)according to the attached manual, by utilizing as primers theoligonucleotide having a nucleotide sequence shown in SEQ ID NO: 346 andthe oligonucleotide having a nucleotide sequence shown in SEQ ID NO:367. An aliquot of the obtained PCR product was utilized as a templatefor a PCR conducted similarly utilizing as primers the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 345 andoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 366.Further, an aliquot of that PCR product was utilized as a template for aPCR conducted similarly utilizing as primers the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 344 and oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 365. The obtainedreaction solution was designated as reaction solution 1.

Further, PCR was conducted with Pyrobest DNA polymerase (Takara ShuzoCompany) according to the attached manual, by utilizing as primers theoligonucleotide having a nucleotide sequence shown in SEQ ID NO: 349 andthe oligonucleotide having a nucleotide sequence shown in SEQ ID NO:364. An aliquot of the obtained PCR product was utilized as a templatefor a PCR conducted similarly utilizing as primers the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 348 andoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 363.Further, an aliquot of that PCR product was utilized as a template for aPCR conducted similarly utilizing as primers the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 347 and oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 362. The obtainedreaction solution was designated as reaction solution 2.

Further, PCR was conducted with Pyrobest DNA polymerase (Takara ShuzoCompany) according to the attached manual by utilizing as primers theoligonucleotide having a nucleotide sequence shown in SEQ ID NO: 352 andoligonucleotide having a nucleotide sequence shown in SEQ JD NO: 361. Analiquot of the obtained PCR product was utilized as a template for a PCRconducted similarly utilizing as primers having the nucleotide sequenceshown in SEQ ID NO: 351 and oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 360. Further, an aliquot of that PCRproduct was utilized as a template for a PCR conducted similarlyutilizing as primers the oligonucleotide having the nucleotide sequenceshown in SEQ ID NO: 350 and oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 359. The obtained reaction solution wasdesignated as reaction solution 3.

Further, PCR was conducted with Pyrobest DNA polymerase (Takara ShuzoCompany) according to the attached manual, by utilizing as primers theoligonucleotide having a nucleotide sequence shown in SEQ ID NO: 355 andoligonucleotide having a nucleotide sequence shown in SEQ ID NO: 358. Analiquot of the obtained PCR product was utilized as a template for a PCRconducted similarly utilizing as primers the oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 354 and oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 357. Further, an aliquot ofthat PCR product was utilized as a template for a PCR conductedsimilarly utilizing as primers the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 353 and oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 356. The obtained reactionsolution was designated as reaction solution 4.

The reaction solutions 1 to 4 obtained in such a way were mixed. PCR wasconducted with Pyrobest DNA polymerase (Takara Shuzo Company) accordingto the attached manual, by utilizing as a template an aliquot of themixture thereof and by utilizing as primers the oligonucleotide having anucleotide sequence shown in SEQ ID NO: 344 and oligonucleotide having anucleotide sequence shown in SEQ ID NO: 356. The nucleotide sequence ofthe amplified DNA was confirmed. There was obtained a DNA having asequence in which the nucleotide sequence 5′-cat-3′ is connectedupstream of the 5′ terminus and the nucleotide sequence 5′-aagctt-3′ isconnected downstream of the 3′ terminus of the nucleotide sequence shownin SEQ ID NO: 368.

The codon usage of the present invention DNA (A23) having the nucleotidesequence shown in SEQ ID NO: 232 (GC content of 73.10%) is shown inTable 28 and Table 29. The codon usage of soybean (GC content of 46.12%)is shown in Table 24 and Table 25. The codon usage of the presentinvention DNA (A23)S having the nucleotide sequence shown in SEQ ID NO:368 (GC content of 52.38%) is shown in Table 30 and Table 31. TABLE 28codon % codon % TTT 0.00 TCT 0.00 TTC 4.01 TCC 1.50 TTA 0.00 TCA 0.00TTG 0.00 TCG 0.50 CTT 0.00 CCT 0.00 CTC 4.26 CCC 5.76 CTA 0.00 CCA 0.00CTG 7.77 CCG 2.26 ATT 0.00 ACT 0.00 ATC 4.51 ACC 3.76 ATA 0.00 ACA 0.00ATG 2.26 ACG 2.76 GTT 0.00 GCT 0.25 GTC 3.51 GCC 9.27 GTA 0.00 GCA 0.75GTG 2.51 GCG 1.75

TABLE 29 codon % codon % TAT 0.00 TGT 0.00 TAC 1.00 TGC 0.75 TAA 0.25TGA 0.00 TAG 0.00 TGG 0.75 CAT 0.00 CGT 0.50 CAC 2.26 CGC 6.02 CAA 0.50CGA 0.25 CAG 2.51 CGG 3.01 AAT 0.00 AGT 0.00 AAC 1.00 AGC 1.25 AAA 0.25AGA 0.00 AAG 0.50 AGG 0.50 GAT 0.00 GGT 0.98 GAC 7.27 GGC 6.27 GAA 1.25GGA 0.25 GAG 5.26 GGG 1.00

TABLE 30 codon % codon % TTT 2.01 TCT 0.75 TTC 2.01 TCC 0.50 TTA 1.00TCA 0.75 TTG 3.01 TCG 0.25 CTT 3.26 CCT 3.01 CTC 2.26 CCC 1.50 CTA 1.00CCA 3.01 CTG 1.50 CCG 0.50 ATT 2.26 ACT 2.26 ATC 1.25 ACC 1.75 ATA 1.00ACA 2.01 ATG 2.26 ACG 0.50 GTT 2.26 GCT 4.51 GTC 1.00 GCC 2.76 GTA 0.75GCA 3.76 GTG 2.01 GCG 1.00

TABLE 31 codon % codon % TAT 0.50 TGT 0.25 TAC 0.50 TGC 0.50 TAA 0.25TGA 0.00 TAG 0.00 TGG 0.75 CAT 1.25 CGT 1.50 CAC 1.00 CGC 1.25 CAA 1.75CGA 0.75 CAG 1.25 CGG 0.50 AAT 0.50 AGT 0.50 AAC 0.50 AGC 0.50 AAA 0.25AGA 3.26 AAG 0.50 AGG 3.01 GAT 4.51 GGT 2.26 GAC 2.76 GGC 1.50 GAA 3.26GGA 2.26 GAG 3.26 GGG 1.50(2) Production of a Transformed E. coli Having the Present InventionProtein (A23)S

The DNA having the nucleotide sequence shown in SEQ ID NO: 368 obtainedin Example 70(1) was digested with restriction enzymes NdeI and HindIII.The obtained DNA and the plasmid pKSN2 digested with NdeI and HindIIIwere ligated to obtain a plasmid in which the DNA having the nucleotidesequence shown in SEQ ID NO: 368 is inserted between the NdeI site andthe HindIII site of pKSN2 (hereinafter referred to as “pKSN1584soy”).Said plasmid was introduced into E. coli JM109. The obtained E. colitransformant was designated JM109/pKSN1584soy.

(3) Expression of the Present Invention Protein (A23)S in E. coli andRecovery of Said Protein

Similarly to Example 4(2), each of E. coli JM109/pKSN1584soy obtained inExample 70(2) and E. coli JM109/pKSN1584F obtained in Example 63(1) wascultured. The cells were recovered. Cell lysate solutions were prepared.Under the method described in Example 4(2), supernatant fractions wereprepared from the cell lysate solutions (hereinafter, the supernatantfraction obtained from E. coli JM109/pKSN1584soy is referred to as “E.coli pKSN1584soy extract” and the supernatant fraction obtained from E.coli JM109/pKSN1584F is referred to as “E. coli pKSN1584F extract”). Theamount of P450 per the protein amount contained in E. coli pKSN1584soyextract was compared to and was higher than the amount of P450 per theprotein amount contained in E. coli pKSN11584F extract.

Example 71 Preparation and Expression of the Present Invention DNA (A25)in Which the Codon Usage has been Adjusted for Expression in Soybean(Hereinafter, Referred to as the “Present Invention DNA (A25)S”)

(1) Preparation of the Present Invention DNA (A25)S

PCR was conducted with Pyrobest DNA polymerase (Takara Shuzo Company)according to the attached manual, by utilizing as primers theoligonucleotide having a nucleotide sequence shown in SEQ ID NO: 371 andthe oligonucleotide having a nucleotide sequence shown in SEQ ID NO:392. An aliquot of the obtained PCR product was utilized as a templatefor a PCR conducted similarly utilizing as primers the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 370 andoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 391.Further, an aliquot of that PCR product was utilized as a template for aPCR conducted similarly utilizing as primers the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 369 and oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 390. The obtainedreaction solution was designated as reaction solution 1.

Further, PCR was conducted with Pyrobest DNA polymerase (Talcara ShuzoCompany) according to the attached manual, by utilizing as primers theoligonucleotide having a nucleotide sequence shown in SEQ ID NO: 374 andthe oligonucleotide having a nucleotide sequence shown in SEQ ID NO:389. An aliquot of the obtained PCR product was utilized as a templatefor a PCR conducted similarly utilizing as primers the oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 373 andoligonucleotide having the nucleotide sequence shown in SEQ ID NO: 383.Further, an aliquot of that PCR product was utilized as a template for aPCR conducted similarly utilizing as primers the oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 372 and oligonucleotidehaving the nucleotide sequence shown in SEQ ID NO: 387. The obtainedreaction solution was designated as reaction solution 2.

Further, PCR was conducted with Pyrobest DNA polymerase (Takara ShuzoCompany) according to the attached manual by utilizing as primers theoligonucleotide having a nucleotide sequence shown in SEQ ID NO: 377 andoligonucleotide having a nucleotide sequence shown in SEQ ID NO: 386. Analiquot of the obtained PCR product was utilized as a template for a PCRconducted similarly utilizing as primers having the nucleotide sequenceshown in SEQ ID NO: 376 and oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 385. Further, an aliquot of that PCRproduct was utilized as a template for a PCR conducted similarlyutilizing as primers the oligonucleotide having the nucleotide sequenceshown in SEQ ID NO: 375 and oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 384. The obtained reaction solution wasdesignated as reaction solution 3.

Further, PCR was conducted with Pyrobest DNA polymerase (Takara ShuzoCompany) according to the attached manual, by utilizing as primers theoligonucleotide having a nucleotide sequence shown in SEQ ID NO: 380 andoligonucleotide having a nucleotide sequence shown in SEQ ID NO: 383. Analiquot of the obtained PCR product was utilized as a template for a PCRconducted similarly utilizing as primers the oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 379 and oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 382. Further, an aliquot ofthat PCR product was utilized as a template for a PCR conductedsimilarly utilizing as primers the oligonucleotide having the nucleotidesequence shown in SEQ ID NO: 378 and oligonucleotide having thenucleotide sequence shown in SEQ ID NO: 381. The obtained reactionsolution was designated as reaction solution 4.

The reaction solutions 1 to 4 obtained in such a way were mixed. PCR wasconducted with Pyrobest DNA polymerase (Takara Shuzo Company) accordingto the attached manual, by utilizing as a template an aliquot of themixture thereof and by utilizing as primers the oligonucleotide having anucleotide sequence shown in SEQ ID NO: 369 and oligonucleotide having anucleotide sequence shown in SEQ ID NO: 381. The nucleotide sequence ofthe amplified DNA was confirmed. There was obtained a DNA having asequence in which the nucleotide sequence 5′-cat-3′ is connectedupstream of the 5′ terminus and the nucleotide sequence 5′-aagctt-3′ isconnected downstream of the 3′ terminus of the nucleotide sequence shownin SEQ ID NO: 393.

The codon usage of the present invention DNA (A25) having the nucleotidesequence shown in SEQ ID NO 234 (GC content of 71.93%) is shown in Table32 and Table 33. The codon usage of soybean (GC content of 46.12%) isshown in Table 24 and Table 25. The codon usage of the present inventionDNA (A25)S having the nucleotide sequence shown in SEQ ID NO: 393 (GCcontent of 52.05%) is shown in Table 34 and Table 35. TABLE 32 codon %codon % TTT 0.00 TCT 0.00 TTC 3.76 TCC 1.25 TTA 0.00 TCA 0.25 TTG 0.00TCG 0.75 CTT 0.00 CCT 0.25 CTC 4.01 CCC 4.01 CTA 0.00 CCA 0.25 CTG 9.52CCG 2.76 ATT 0.00 ACT 0.25 ATC 4.26 ACC 4.01 ATA 0.25 ACA 0.00 ATG 2.26ACG 1.75 GTT 0.00 GCT 0.00 GTC 3.01 GCC 8.52 GTA 0.00 GCA 0.50 GTG 2.51GCG 3.01

TABLE 33 codon % codon % TAT 0.00 TGT 0.25 TAC 1.25 TGC 0.50 TAA 0.25TGA 0.00 TAG 0.00 TGG 1.00 CAT 0.25 CGT 0.75 CAC 2.26 CGC 5.51 CAA 0.00CGA 1.25 CAG 3.01 CGG 3.26 AAT 0.00 AGT 0.00 AAC 1.00 AGC 1.00 AAA 0.25AGA 0.25 AAG 1.00 AGG 0.00 GAT 0.00 GGT 0.25 GAC 7.52 GGC 4.76 GAA 1.00GGA 0.25 GAG 4.76 GGG 1.25

TABLE 34 codon % codon % TTT 1.75 TCT 1.25 TTC 2.01 TCC 0.50 TTA 1.25TCA 0.50 TTG 3.26 TCG 0.00 CTT 3.51 CCT 2.76 CTC 2.51 CCC 1.25 CTA 1.25CCA 2.76 CTG 1.75 CCG 0.50 ATT 2.26 ACT 2.01 ATC 1.25 ACC 1.75 ATA 1.00ACA 1.75 ATG 2.26 ACG 0.50 GTT 2.26 GCT 4.51 GTC 1.00 GCC 2.76 GTA 0.50GCA 3.76 GTG 1.75 GCG 1.00

TABLE 35 codon % codon % TAT 0.50 TGT 0.25 TAC 0.75 TGC 0.50 TAA 0.25TGA 0.00 TAG 0.00 TGG 1.00 CAT 1.25 CGT 1.75 CAC 1.25 CGC 1.50 CAA 1.50CGA 0.75 CAG 1.50 CGG 0.75 AAT 0.50 AGT 0.50 AAC 0.50 AGC 0.50 AAA 0.50AGA 3.26 AAG 0.75 AGG 3.01 GAT 4.76 GGT 2.01 GAC 2.76 GGC 1.25 GAA 2.76GGA 2.01 GAG 3.01 GGG 1.25(2) Production of a Transformed E. coli Having the Present InventionProtein (A2)S

The DNA having the nucleotide sequence shown in SEQ ID NO: 393 obtainedin Example 71(1) was digested with restriction enzymes NdeI and HindIII.The obtained DNA and the plasmid pKSN2 digested with NdeI and HindIIIwere ligated to obtain a plasmid in which the DNA having the nucleotidesequence shown in SEQ ID NO: 393 is inserted between the NdeI site andthe HindIII site of pKSN2 (hereinafter referred to as “pKSN1609soy”).Said plasmid was introduced into E. coli JM109. The obtained E. colitransformant was designated JM109/pKSN1609soy.

(3) Expression of the Present Invention Protein (A25)S in E. coli andRecovery of said Protein

Similarly to Example 4(2), each of E. coli JM109/pKSN1609soy obtained inExample 71(2) and E. coli 3M109/pKSN1609F obtained in Example 67(1) wascultured. The cells were recovered. Cell lysate solutions were prepared.Under the method described in Example 4(2), supernatant fractions wereprepared from the cell lysate solutions (hereinafter, the supernatantfraction obtained from E. coli JM109/pKSN1609soy is referred to as “E.coli pKSN1609soy extract” and the supernatant fraction obtained from E.coli JM109/pKSN1609F is referred to as “E. coli pKSN1609F extract”). Theamount of P450 per the protein amount contained in E. coli pKSN1609soyextract was compared to and was higher than the amount of P450 per theprotein amount contained in E. coli pKSN1609F extract.

Example 72 Preparation of the Present Invention Antibody (A) Recognizingthe Present Invention Protein (A25) (Hereinafter Referred to as “PresentInvention Antibody (A25)”)

(1) Preparation of the Extract of an E. coli Expressing the PresentInvention Protein (A25)

In accordance with the method described in Example 4(2), E. coliJM109/pKSN1609soy, produced in Example 71(2), was pre-culturedovernight. The obtained cultured medium was inoculated to 1 L of TBmedium containing 50 μg/ml of ampicillin and cultured at 26° C. Then5-aminolevulinic acid was added to the final concentration of 500 μM,and IPTG was added to a final concentration of 1 mM, and that wasfurther cultured. The cells were recovered from the cultured medium,were washed with 0.05M Tris-HCl Buffer (pH7.5) and then suspended in 100ml of said buffer containing 1 mM PMSF. The obtained cell culture mediumwas subjected 3 times to a sonicator (Sonifier (Branson Sonic PowerCompany)) at 10 minutes each under the conditions of output 5, dutycycle 30%, in order to obtain cell lysate solutions. After centrifugingthe cell lysate solutions (9,000×g, 10 minutes) the supernatants wererecovered and centrifuged (200,000×g, 70 minutes) to recover supernatantfractions (hereinafter, the supernatant fraction obtained from E. coliJM109/pKSN1609soy is referred to as “E. coli pKSN1609soy extract”.

(2) Purification of the Present Invention Protein (A25)

The supernatant fraction obtained in Example 72(1) (E. coli pKSN1609soyextract) was injected into a Hiload HiLoad 16/10 Q Sepharose HP column(Amersham Bioscience Company). Next, after flowing 40 ml of 20 mMbistrispropane buffer (pH7.0) into the column, 20 mM bistrispropanebuffer was flown with a linear gradient of NaCl (gradient of NaCl was0.00125M/minute, range of NaCl concentration was from 0M to 0.375M, flowrate was 3 ml/minute) to fraction recover 10 ml of fractions eluting atthe NaCl concentration of from 0.088M to 0.100M.

The recovered fractions were subjected to a PD-10 column (AmershamBiosciences Company) and eluted with 20 mM bistrispropane buffer (pH7.0)to recover the fractions containing protein. Next, said fractions wereinjected into a MonoQ HR 10/10 (Amersham Biosciences Company). Sixteenmilliliters (16 ml) of 20 mM bistrispropane buffer was flown into thecolumn. Next, 20 mM bistrispropane buffer was flown with a lineargradient of NaCl (gradient of NaCl was 0.001042M/minute, range of NaClconcentration was from 0M to 0.25M, flow rate was 4 ml/minute) tofraction recover 8 ml of fractions eluting at the NaCl concentration offrom 0.060M to 0.069M.

The recovered fractions were diluted 2.5 fold with 20 mM bistispropanebuffer (pH7.0) and injected into a MonoQ HR 5/5 column (AmershamBiosciences Company). Next, after flowing 2 ml of 20 mM bistrispropanebuffer (pH7.0) into the column, 20 mM bistrispropane buffer was flownwith a linear gradient of NaCl (gradient of NaCl was 0.008333M/minute,range of NaCl concentration was from 0M to 0.25K flow rate was 1ml/minute) to fraction recover 0.5 ml of fractions eluting at the, NaClconcentration of from 0.073M to 0.077M.

The fractions purified in such a way were analyzed with SDS-PAGE byutilizing a “PAG mini Daiichi 10/20” (Daiichi Pure Chemicals Co., Ltd.)to confirm that those fractions were fractions which mainly contain thepresent invention protein (A25).

(3) Preparation of the Present Invention Antibody (A25)

Preparation of the present invention antibody was conducted accordinglyto the method described in Example 44(3). However, instead of utilizingthe present invention protein (A1), the present invention protein (A25)obtained in Example 72(2) was utilized to obtain antiserum containingthe present invention antibody (A25).

Example 73 Detection of the Present Invention Protein by the PresentInvention Antibody (A25)

An immunoblot was conducted by utilizing the present invention antibody(A25) obtained in Example 72(3) with each of the E. coli extracts. Therewas a SDS polyacrylamide electrophoresis (400 mA, 1 hour) of: the E.coli pKSN452F extract obtained in Example 49(2) (containing about 2 pmolof the present invention protein (A16)); the E. coli pKSN60SF extractobtained in Example 51(2) (containing about 2 pmol of the presentinvention protein (A17)); the E. coli pKSN646BF extract obtained inExample 53(2) (containing about 2 pmol of the present invention protein(A18)); the E. coli pKSN1502F extract obtained in Example 55(2)(containing about 2 pmol of the present invention protein (A19)); the E.coli pKSN1525F extract obtained in Example 57(2) (containing about 2pmol of the present invention protein (A20)); the E. coli pKSN1543BFextract obtained in Example 59(2) (containing about 2 pmol of thepresent invention protein (A21)); the E. coli pKSN1558SF extractobtained in Example 61(2) (containing about 2 pmol of the presentinvention protein (A22)); the E. coli pKSN1584F extract obtained inExample 63(2) (containing about 2 pmol of the present invention protein(A23)); the E. coli pKSN1589BF extract obtained in Example 65(2)(containing about 2 pmol of the present invention protein (A24)); the E.coli pKSN1609F extract obtained in Example 67(2) (containing about 0.5pmol of the present invention protein (A25)); the E. coli pKSN1584soyextract obtained in Example 70(3) (containing about 2 pmol of thepresent invention protein (A23)); the E. coli pKSN1609soy extractobtained in Example 71(3) (containing about 0.5 pmol of the presentinvention protein (A25)); and the E. coli pKSN2 extract obtained inExample 67(2) (containing about 0.8 mg of protein). The proteins in saidgel were transferred to a PVDF membrane according to the methoddescribed in Example 45. The PDVF membrane obtained in Example 45(hereinafter referred to as “PDVF membrane (A)”) and the PDVF membraneobtained from the above method (hereinafter referred to as “PDVFmembrane (B)”) were reacted with the antiserum obtained in Example72(3), according to the method described Example 45. Subsequently, therewas conducted a reaction with the secondary antibody, a washing and astaining in accordance with the method described in Example 45. Stainsfor bands corresponding to the present invention proteins (A1), (A2),(A3), (A4), (A11), (A12), (A13), (A14) and (A15) as well as the presentproteins (A9) and (A10) were detected on the PDVF membrane (A). Stainsfor bands corresponding to the present invention proteins (A16), (A17),(A18), (A19), (A20), (A21), (A22), (A23), (A24) and (A25) were detectedon the PDVF membrane (B). No stained band was detected with the reagentof E. coli pKSN2 extract obtained in Example 4(2) (containing 0.78 mg ofprotein) of PVDF membrane (A) and with the reagent of E. coli pKSN2extract obtained in Example 67(2) (containing 0.8 mg of protein) of PVDFmembrane (B).

Example 74 Introduction of the Present Invention DNA (A23)S into a Plant

(1) Construction of a Chloroplast Expression Plasmid Containing thePresent Invention DNA (A23)S for Direct Introduction—Part 1

A plasmid containing a chimeric DNA in which the present invention DNA(A23)S was connected immediately after the nucleotide sequence encodingthe chloroplast transit peptide of soybean (cv. Jack) RuBPC smallsubunit without a change of frames in the codons was constructed as aplasmid for introducing the present invention DNA (A23)S into a plantwith the particle gun method.

First, DNA comprising the nucleotide sequence shown in SEQ ID NO: 398was amplified by PCR. The PCR was conducted by utilizing as a templatepKSN1584soy obtained in Example 70(2) and by utilizing as primers anoligonucleotide consisting of the nucleotide sequence shown in SEQ IDNO: 397 and an oligonucleotide consisting of the nucleotide sequenceshown in SEQ ID NO: 398. The PCR utilized KOD-plus (Toyobo Company). ThePCR carried out after conducting once a maintenance at 94° C. for 2minutes; 20 cycles of a cycle that included maintaining 94° C. for 30seconds, followed by 53° C. for 30 seconds, and followed by 68° C. for90 seconds; and a final maintenance at 68° C. for 3 minutes. Theamplified DNA was recovered and purified with MagExtractor-PCR &Gel-Clean up (Toyobo Company) by conducting the procedures according tothe attached manual. By treating the obtained DNA with TaKaRa BKLKit(Takara Shuzo Company) according to the attached manual, the DNA wasblunt ended and had the 5′ terminus phosphorylated A DNA comprising anucleotide sequence shown in SEQ ID NO: 368 was recovered. Afterdigesting plasmid pUC19 (Takara Shuzo Company) with SmaI, the 5′terminus was dephosphorylated with calf intestine alkaline phosphatase(Takara Shuzo Company). A plasmid was produced by ligating the resultingdephosphorylmated DNA and the DNA comprising the nucleotide sequenceshown in SEQ ID NO: 368. After digesting the obtained plasmid withrestriction enzymes EcoT221 and SacI, the DNA comprising the nucleotidesequence shown in SEQ ID NO: 368 was recovered. After digesting plasmidpUCrSt657 obtained in Example 16(2) with restriction enzymes EcoT221 andSacI, there was isolated a DNA of about 2.9 kbp having a nucleotidesequence derived from pUC19 and a sequence encoding a chloroplasttransit peptide of soybean (cv. Jack) RuBPC small subunit. The obtainedDNA and the above DNA comprising the nucleotide sequence shown in SEQ IDNO: 368 were ligated to obtain pUCrSt1584soy (FIG. 54) containing achimeric DNA in which the present invention DNA (A23)S was connectedimmediately after the nucleotide sequence encoding the chloroplasttransit peptide of soybean (cv. Jack) RuBPC small subunit without achange of flames in the codons.

The obtained plasmid pUCrSt1584soy was digested with restriction enzymesBamHI and SacI to isolate a DNA comprising a nucleotide sequence shownin SEQ ID NO: 368. Said DNA was inserted between the BglII restrictionsite and the SacI restriction site of plasmid pNdG6-ΔT obtained inExample 16(2) to obtain plasmid pSUM-NdG6-rSt-1584soy (FIG. 55) whereinthe CR16G6 promoter has connected downstream the chimeric DNA in whichsaid DNA was connected immediately after the nucleotide sequenceencoding the chloroplast transit peptide of soybean (cv. Jack) RuBPCsmall subunit without a change of frames in the codons.

Next, the plasmid was introduced into E. coli DH5α competent cells(Takara Shuzo Company) and the ampicillin resistant cells were selected.Further, the nucleotide sequences of the plasmids contained in theampicillin resistant strains were determined by utilizing BigDyeTerminator Cycle Sequencing Ready Reaction kit v3.0 (PE AppliedBiosystems Company) and DNA sequencer 3100 (PE Applied BiosytemsCompany). As a result, it was confirmed that plasmidpSUM-NdG6-rSt-1584soy has the nucleotide sequence shown in SEQ ID NO:368.

(2) Construction of a Chloroplast Expression Plasmid Having the PresentInvention DNA (A23)S for Direct Introduction—Part (2)

A plasmid was constructed for introducing the present invention DNA(A23)S into a plant with the particle gun method. The plasmid containeda chimeric DNA in which the present invention DNA (A23)S was connectedimmediately after the nucleotide sequences encoding the chloroplasttransit peptide of soybean (cv. Jack) RuBPC small subunit and encodingthereafter 12 amino acids of the mature protein, without a change offrames in the codons. First, DNA comprising the nucleotide sequenceshown in SEQ ID NO; 368 was amplified by PCR The PCR was conducted byutilizing as a template pKSN1584soy obtained in Example 70 and byutilizing as primers an oligonucleotide consisting of the nucleotidesequence shown in SEQ ID NO: 399 and an oligonucleotide consisting ofthe nucleotide sequence shown in SEQ ID NO; 398. The PCR utilizedKOD-plus (Toyobo Company). The PCR carried out after conducting once amaintenance at 94° C. for 2 minutes; 25 cycles of a cycle that includedmaintaining 94° C. for 30 seconds, followed by 46° C. for 30 seconds,and followed by 68° C. for 90 seconds; and a final maintenance at 68° C.for 3 minutes. The amplified DNA was recovered and purified withMagExtractor-PCR & Gel-Clean up (Toyobo Company) by conducting theprocedures according to the attached manual. By treating the obtainedDNA with TaKaRa BKLKit (Takara Shuzo Company) according to the attachedmanual, the DNA was blunt ended and had the 5′ terminus phosphorylated.A DNA comprising a nucleotide sequence shown in SEQ ID NO: 368 wasrecovered. After digesting plasmid pKF19ΔBs obtained in Example 15(3)with SmaI, the 5′ terminus was dephosphorylated with calf intestinealkaline phosphatase (Takara Shuzo Company). A plasmid was produced byligating the resulting dephosphorylated DNA and the DNA comprising thenucleotide sequence shown in SEQ ID NO: 368. After digesting theobtained plasmid with restriction enzymes BspHI and SacI, the DNAcomprising the nucleotide sequence shown in SEQ ID NO: 368 wasrecovered. Next, plasmid pKFrSt12-657 obtained in Example 16(3) wasdigested with restriction enzymes BspHI and SacI to isolate the DNAderived from plasmid pKFrSt12. Said DNA was ligated with the DNA whichwas digested with restriction enzymes SacI and BspHI and which comprisesthe nucleotide sequence shown in SEQ ID NO: 368, in order to obtainplasmid pKFrSt12-1584soy (FIG. 56) containing the chimeric DNA in whichthe present invention DNA (A23)S was connected immediately after thenucleotide sequences encoding the chloroplast transit peptide of soybean(cv. Jack) RuBPC small subunit and encoding thereafter 12 amino acids ofthe mature protein, without a change of frames in the codons.

The obtained plasmid pKFrSt12-1584soy was digested with restrictionenzymes BamHI and SacI to isolate the DNA comprising the nucleotidesequence shown in SEQ ID NO: 368. Said DNA was inserted between theBglII restriction site and the SacI restriction site of plasmid pNdG6-ΔTto obtain plasmid pSUM-NdG6-rSt12-1584soy (FIG. 57) wherein the CR16G6promoter has connected downstream the chimeric DNA in which said DNA wasconnected immediately after the nucleotide sequence encoding thechloroplast transit peptide of soybean (cv. Jack) RuBPC small subunitwithout a change of frames in the codons.

Next, the plasmid was introduced into E. coli DH5α competent cells(Takara Shuzo Company) and the ampicillin resistant cells were selected.Further, the nucleotide sequences of the plasmids contained in theampicillin resistant strains were determined by utilizing BigDyeTerminator Cycle Sequencing Ready Reaction kit v3.0 (PE AppliedBiosystems Company) and DNA sequencer 3100 (PE Applied BiosytemsCompany). As a result, it was confirmed that plasmidpSUM-NdG6-rSt12-1584soy has the nucleotide sequence shown in SEQ ID NO:368.

(3) Introduction of the Present Invention DNA (A23)S into Soybean

The globular embryos of soybeans (cultivar: Fayette and lack) wereprepared according to the method described in Example 47(3).

The obtained globular embryo was transplanted into fresh somatic embryogrowth medium and cultured for 2 to 3 days. The plasmidpSUM-NdG6-rSt-1584soy produced in Example 74(1) or the plasmidpSUM-NdG6-rSt12-1584soy produced in Example 74(2) were introduced intothose globular embryos according to the method described in Example17(2).

(4) Selection of Somatic Embryo with Hygromycin

Selection by hygromycin of a spherica-typel embryo after theintroduction of the gene, obtained in Example 74(3), is conductedaccording to the method described in Example 47(4).

(5) Selection of Somatic Embryo with Compound (II)

Selection by compound (II) of a globular embryo after the introductionof the gene, obtained in Example 74(3), is conducted according to themethod described in Example 47(5).

(6) Plant Regeneration from the Somatic Embryo, Acclimation andCultivation

In accordance with the method described in Example 47(6), the plantregeneration is conducted from the globular embryos selected in Examples74(4) or 74(5).

The plant with roots and developed leaves undergo the acclimation andcultivation accordingly with the method described in Example 17(6) andare harvested.

(7) Evaluation of the Resistance to Herbicidal Compound (II)

The degree of resistance against compound (II) of the regenerated plantobtained in Example 74(6) is evaluated in accordance with the methoddescribed in Example 17(4).

(8) Construction of a Chloroplast Expression Plasmid Having the PresentInvention DNA (A23)S for Agrobacterium Introduction

A plasmid for introducing the present invention DNA (A23)S into a plantwith the agrobacterium method is constructed. PlasmidpSUM-NdG6-rSt-1584soy was digested with restriction enzymes HindIII andEcoRI, to isolate the chimeric DNA in which the present invention DNA(A23)S was connected immediately after the nucleotide sequence encodingthe chloroplast transit peptide of soybean (cv. Jack) RuBPC smallsubunit without a change of frames in the codons. Said DNA was insertedinto between the HindIII restriction site and the EcoRI restriction siteof the above binary plasmid vector pBI121 S obtained in Example 18 toobtain plasmid pBI-NdG6-rSt-1584soy (FIG. 58). Further, plasmidpSUM-NdG6-rSt12-1584soy was digested with restriction enzyme NotI, toisolate a chimeric DNA in which the present invention DNA (A23)S wasconnected immediately after the nucleotide sequences encoding thechloroplast transit peptide of soybean (cv. Jack) RuBPC small subunitand encoding thereafter 12 amino acids of the mature protein, without achange of frames in the codons. Such a DNA was inserted between theHindIII restriction site and EcoRI restriction site of the above binaryplasmid vector pBI121S to obtain plasmids pBI-NdG6-rSt12-1584soy (FIG.59).

(9) Introduction of the Present Invention DNA (A23)S to Tobacco

The present invention DNA (A23)S was introduced into tobacco with theagrobacterium method, utilizing plasmid pBI-NdG6-rSt-1584soy andpBI-NdG6rSt12-1584soy obtained in Example 74(8).

First, in accordance with the method described in Example 19, each ofthe plasmids pBI-NdG6-rSt-1584soy and pBI-NdG6-rSt12-1584soy wasintroduced into Agrobacterium tumefaciens LBA4404 (Clontech Company).Each of the transgenic agrobacterium bearing pBI-NdG6rSt-1584soy orpBI-NdG6-rSt12-1584soy were isolated.

Next, said agrobacterium bearing the plasmids are utilized to introducegenes into tobacco according to the method described in Example 47(9) toobtain, respectively, transgenic tobaccos which have incorporated theT-DNA region of pBI-NdG6-rSt-1584soy or pBI-NdG6-rSt12-1584soy.

(10) Evaluation of the Resistance Utilizing a Leaf Piece of the PresentInvention DNA (A23)S Transgenic Tobacco

Leaves are taken from 35 transgenic tobaccos obtained in Example 74(9).Each leaf is divided into pieces in which each piece is 5 to 7 mm wide.Leaf pieces are planted onto MS agar medium containing compound (II) orcompound (XII) and cultured in the light at room temperature. Afterseveral days of culturing, the herbicidal damage of each of the leafpieces is observed. As a control, leaf pieces of wild type tobacco areutilized. The resistance of the transgenic tobacco is evaluated byscoring the leaf pieces which continuously grow, leaf pieces which havechemical damage, and leaf pieces which tamed white and have withered.

Example 75 Introduction of the Present Invention DNA (A25)S into a Plant

(1) Construction of a Chloroplast Expression Plasmid Containing thePresent Invention DNA (A25)S for Direct Introduction—Part 1

A plasmid containing a chimeric DNA in which the present invention DNA(A25)S was connected immediately after the nucleotide sequence encodingthe chloroplast transit peptide of soybean (cv. Jack) RuBPC smallsubunit without a change of frames in the codons was constructed as aplasmid for introducing the present invention DNA (A25)S into a plantwith the particle gun method.

First, DNA comprising the nucleotide sequence shown in SEQ ID NO: 393was amplified by PCR. The PCR was conducted by utilizing as a templatepKSN1609soy obtained in Example 71(2) and by utilizing as primers anoligonucleotide consisting of the nucleotide sequence shown in SEQ IDNO: 400 and an oligonucleotide consisting of the nucleotide sequenceshown in SEQ ID NO: 401. The PCR utilized KOD-plus (Toyobo Company). ThePCR carried out after conducting once a maintenance at 94° C. for 2minutes; 20 cycles of a cycle that included maintaining 94° C. for 30seconds, followed by 53° C. for 30 seconds, and followed by 68° C. for90 seconds; and a final maintenance at 68° C. for 3 minutes, Theamplified DNA was recovered and purified with MagExtractor-PCR &Gel-Clean up (Toyobo Company) by conducting the procedures according tothe attached manual. By treating the obtained DNA with TaKaRa BKLKit(Takara Shuzo Company) according to the attached manual, the DNA wasblunt ended and had the 5′ terminus phosphorylated. A DNA comprising anucleotide sequence shown in SEQ ID NO: 393 was recovered. Afterdigesting plasmid pUC19 (Takara Shuzo Company) with SmaI, the 5′terminus was dephosphorylated with calf intestine alkaline phosphatase(Takara Shuzo Company). A plasmid was produced by ligating the resultingdephosphorylated DNA and the DNA comprising the nucleotide sequenceshown in SEQ ID NO: 393. After digesting the obtained plasmid withrestriction enzymes EcoT221 and SacI, the DNA comprising the nucleotidesequence shown in SEQ ID NO: 393 was recovered. After digesting plasmidpUCrSt657 obtained in Example 16(2) with restriction enzymes EcoT221 andSacI, there was isolated a DNA of about 2.9 kbp having a nucleotidesequence derived from pUC19 and a sequence encoding a chloroplasttransit peptide of soybean (cv. Jack) RuBPC small subunit The obtainedDNA and the above DNA comprising the nucleotide sequence shown in SEQ IDNO: 393 were ligated to obtain pUCrSt1609soy (FIG. 60) containing achimeric DNA in which the present invention DNA (A25)S was connectedimmediately after the nucleotide sequence encoding the chloroplasttransit peptide of soybean (cv. Jack) RuBPC small subunit without achange of frames in the codons.

The obtained plasmid pUCrSt1609soy was digested with restriction enzymesBamHI and SacI to isolate a DNA comprising a nucleotide sequence shownin SEQ ID NO: 393. Said DNA was inserted between the BglII restrictionsite and the SacI restriction site of plasmid pNdG6-ΔT to obtain plasmidpSUM-NdG6-rSt-1609soy (FIG. 61) wherein the CR16G6 promoter hasconnected downstream the chimeric DNA in which said DNA was connectedimmediately after the nucleotide sequence encoding the chloroplasttransit peptide of soybean (cv. Jack) RuBPC small subunit without achange of frames in the codons.

Next, the plasmid was introduced into E. coli DH5α competent cells(Takara Shuzo Company) and the ampicillin resistant cells were selected.Further, the nucleotide sequences of the plasmids contained in theampicillin resistant strains were determined by utilizing BigDyeTerminator Cycle Sequencing Ready Reaction kit v3.0 (PE AppliedBiosystems Company) and DNA sequencer 3100 (PE Applied BiosytemsCompany). As a result, it was confirmed that plasmidpSUM-NdG6-rSt-1609soy has the nucleotide sequence shown in SEQ ID NO:393.

(2) Construction of a Chloroplast Expression Plasmid Having the PresentInvention DNA (A25)S for Direct Introduction—Part (2)

A plasmid was constructed for introducing the present invention DNA(A25)S into a plant with the particle gun method. The plasmid containeda chimeric DNA in which the present invention DNA (A25)S was connectedimmediately after the nucleotide sequences encoding the chloroplasttransit peptide of soybean (cv. Jack) RuBPC small subunit and encodingthereafter 12 amino acids of the mature protein, without a change offrames in the codons. First, plasmid pUCrSt1609soy obtained in Example75(1) has inserted into its EcoT221 restriction site, the linkerEcoT221-12aa-EcoT221 (FIG. 62) obtained by annealing the oligonucleotideconsisting of the nucleotide sequence shown in SEQ ID NO: 402 and theoligonucleotide consisting of the nucleotide sequence shown in SEQ IDNO: 403. There was obtained plasmid pUCrSt12-1609soy (FIG. 63)containing the chimeric DNA in which the present invention DNA (A25)Swas connected immediately after the nucleotide sequences encoding thechloroplast transit peptide of soybean (cv. Jack) RuBPC small subunitand encoding thereafter 12 amino acids of the mature protein, without achange of frames in the codons.

The obtained plasmid pUCrSt12-1609soy was digested with restrictionenzymes BamHI and SacI to isolate the DNA comprising the nucleotidesequence shown in SEQ ID NO; 393. Said DNA was inserted between theBglII restriction site and the SacI restriction site of plasmidpNdG6-ΔT, obtained in Example 16(2), to obtain plasmidpSUM-NdG6-rSt12-1609soy (FIG. 64) wherein the CR16G6 promoter hasconnected downstream the chimeric DNA in which said DNA was connectedimmediately after the nucleotide sequence encoding the chloroplasttransit peptide of soybean (cv. Jack) RuBPC small subunit without achange of frames in the codons.

Next, the plasmid was introduced into E. coli DH5α competent cells(Takara Shuzo Company) and the ampicillin resistant cells were selected.Further, the nucleotide sequences of the plasmids contained in theampicillin resistant strains were determined by utilizing BigDyeTerminator Cycle Sequencing Ready Reaction kit v3.0 (PE AppliedBiosystems Company) and DNA sequencer 300 (PE Applied BiosytemsCompany). As a result, it was confirmed that plasmidpSUM-NdG6-rSt12-1609soy has the nucleotide sequence shown in SEQ ID NO:393.

(3) Introduction of the Present Invention DNA (A23)S into soybean

The globular embryos of soybeans (cultivar: Fayette and Jack) wereprepared according to the method described in Example 47(3).

The obtained globular embryo was transplanted into fresh somatic embryogrowth medium and cultured for 2 to 3 days. The plasmidpSUM-NdG6-rSt-1609soy produced in Example 75(1) or the plasmidpSUM-NdG6-rSt12-1609soy produced in Example 75(2) were introduced intothose globular embryos according to the method described in Example17(2).

(4) Selection of Somatic Embryo with Hygromycin

Selection by hygromycin of a globular embryo after the introduction ofthe gene, obtained in Example 75(3), is conducted according to themethod described in Example 2547(4).

(5) Selection of Somatic Embryo with Compound (II)

Selection by compound (II) of a globular embryo after the introductionof the gene, obtained in Example 75(3), is conducted according to themethod described in Example 47(5).

(6) Plant Regeneration from the Somatic Embryo, Acclimation andCultivation

In accordance with the method described in Example 47(6), the plantregeneration is conducted from the globular embryos selected in Examples74(4) or 74(5).

The plant with roots and developed leaves undergo the acclimation andcultivation accordingly with the method described in Example 17(6) andare harvested.

(7) Evaluation of the Resistance to Herbicidal Compound (I)

The degree of resistance against compound (II) of the regenerated plantobtained in Example 75(6) is evaluated in accordance with the methoddescribed in Example 17(4).

(8) Construction of a Chloroplast Expression Plasmid Having the PresentInvention DNA (A25)S for Agrobacterium Introduction

A plasmid for introducing the present invention DNA (A25)S into a plantwith the agrobacterium method is constructed. Plasmid pSUM-NdG6rSt-I609soy was digested with restriction enzymes HindIII and EcoRI, toisolate the chimeric DNA in which the present invention DNA (A25)S wasconnected immediately after the nucleotide sequence encoding thechloroplast transit peptide of soybean (cv. Jack) RuBPC small subunitwithout a change of frames in the codons. Said DNA was inserted intobetween the HindIII restriction site and the EcoRI restriction site ofthe binary plasmid vector pB1121S obtained in Example 18 to obtainplasmid pBI-NdG6-rSt-1609soy (FIG. 65), Further, plasmidpSUM-NdG6-rSt12-1609soy was digested with restriction enzyme NotI, toisolate a chimeric DNA in which the present invention DNA (A25)S wasconnected immediately after the nucleotide sequences encoding thechloroplast transit peptide of soybean (cv. Jack) RuBPC small subunitand encoding thereafter 12 amino acids of the mature protein, without achange of frames in the codons. Such a DNA was inserted between theHindIII restriction site and EcoRI restriction site of the above binaryplasmid vector pBI121S to obtain plasmids pBI-NdG6-rSt12-1609soy (FIG.66).

(9) Introduction of the Present Invention DNA (A23)S to Tobacco

The present invention DNA (A25)S was introduced into tobacco with theagrobacterium method, utilizing plasmid pBI-NdG6-rSt-1609soy andpB3-NdG6rSt12-1609soy obtained in Example 75(8).

First, in accordance with the method described in Example 19, each ofthe plasmids pBI-NdG6-rSt-1609soy and pBI-NdG6-rSt12-1609soy wasintroduced into Agrobacterium tumefaciens LBA4404 (Clontech Company).Each of the transgenic agrobacterium bearing pBI-NdG6-rSt-1609soy orpBI-NdG6-rSt12-1609soy were isolated.

Next, said agrobacterium bearing the plasmids are utilized to introducegenes into tobacco according to the method described in Example 47(9) toobtain, respectively, transgenic tobaccos which have incorporated theT-DNA region of pBI-NdG6-rSt-1609soy or pBI-NdG6-rSt12-1609soy.

(10) Evaluation of the Resistance Utilizing a Leaf Piece of the PresentInvention DNA (A25)S Transgenic Tobacco

Leaves are taken from the transgenic tobaccos obtained in Example 75(9).Such leaves are utilized to evaluate the resistance of the transgenictobacco against compound (II) or compound (XII) according to the methodof Example 74(10).

APPLICABILITY TO INDUSTRY

With the present invention, it is possible to provide a protein havingthe ability to metabolize a PPO inhibiting herbicidal compound and toconvert such a compound to a compound of lower herbicidal activity; aDNA encoding such a protein; and a plant resistant to a herbicidalcompound expressing such a protein.

Sequence Free Text

-   SEQ ID NO: 35-   Designed oligonucleotide primer for PCR-   SEQ ID NO:36-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 37-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 38-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 39-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 40-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 41-   Designed oligonucleotide primer for PCR-   SEQ ID NO; 42-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 43-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 44-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 45-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 46-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 47-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 48-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 49-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 50-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 51-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 52-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 53-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 54-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 55-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 56-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 57-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 58-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 59-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 60-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 61-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 62-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 63-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 64-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 65-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 66-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 67-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 68-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 70-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 71-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 72-   Designed oligonucleotide primer for PCR-   SEQ ID NO:73-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 74-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 75-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 76-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 77-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 79-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 80-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 81-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 82-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 83-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 86-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 87-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 89-   Designed oligonucleotide linker for construction of expression    vector-   SEQ ID NO: 90-   Designed oligonucleotide linker for construction of expression    vector-   SEQ ID NO: 91-   Designed oligonucleotide linker for construction of expression    vector-   SEQ ID NO: 92-   Designed oligonucleotide linker for construction of expression    vector-   SEQ ID NO: 93-   Designed oligonucleotide primer for PCR-   SEQ ID NO 94-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 95-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 96-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 97-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 98-   Designed oligonucleotide linker for construction of expression    vector-   SEQ ID NO: 99-   Designed oligonucleotide linker for construction of expression    vector-   SEQ ID NO: 100-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 101-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 102-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 103-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 104-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 105-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 106-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 107-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 114-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 115-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 116-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 117-   Designed oligonucleotide primer for PCR-   SEQ ID NO 118-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 119-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 120-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 121-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 122-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 123-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 124-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 125-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 126-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 127-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 128-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 129-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 130-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 131-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 132-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 133-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 134-   Designed oligonucleotide linker for construction of expression    vector-   SEQ ID NO: 135-   Designed oligonucleotide linker for construction of expression    vector-   SEQ ID NO: 161-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 162-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 163-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 164-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 165-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 166-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 167-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 168-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 169-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 170-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 171-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 172-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 173-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 174-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 175-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 176-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 177-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 178-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 179-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 180-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 181-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 182-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 183-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 184-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 185-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 186-   Designed oligonucleotide primer for DNA sequencing-   SEQ ID NO: 187-   Designed oligonucleotide primer for DNA sequencing-   SEQ ID NO: 188-   Designed oligonucleotide primer for DNA sequencing-   SEQ ID NO: 189-   Designed oligonucleotide primer for DNA sequencing-   SEQ ID NO: 190-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 191-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 192-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 193-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 194-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 195-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 196-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 197-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 198-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 199-   Designed oligonucleotide primer for PCR-   SEQ ID NO; 200-   Designed oligonucleotide primer for PCR-   SEQ ID NO:201-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 202-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 203-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 204-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 205-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 206-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 207-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 208-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 209-   Designed oligonucleotide primer for PCR-   SEQ ID NO:210-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 211-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 212-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 213-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 214-   Designed polynucleotide encoding amino acid sequence of SEQ ID No. 1-   SEQ ID NO: 265-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 266-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 267-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 268-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 269-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 270-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 271-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 272-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 273-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 274-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 275-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 276-   Designed oligonucleotide primer for DNA sequencing-   SEQ ID NO: 277-   Designed oligonucleotide primer for DNA sequencing-   SEQ ID NO: 278-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 279-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 280-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 281-   Designed oligonucleotide primer for DNA sequencing-   SEQ ID NO: 282-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 283-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 284-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 285-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 286-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 287-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 288-   Designed oligonucleotide primer for DNA sequencing-   SEQ ID NO: 289-   Designed oligonucleotide primer for PCR-   SEQ ID NO 290-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 291-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 292-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 293-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 294-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 295-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 296-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 297-   Designed oligonucleotide primer for PCR-   SEQ ID NO:298-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 299-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 300-   Designed oligonucleotide primer for DNA sequencing-   SEQ ID NO 301-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 302-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 303-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 304-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 305-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 306-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 307-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 308-   Designed oligonucleotide primer for DNA sequencing-   SEQ ID NO: 309-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 310-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 311-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 312-   Designed oligonucleotide primer for PCR-   SEQ ID NO 313-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 314-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 315-   Designed oligonucleotide primer for DNA sequencing-   SEQ ID NO: 316-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 317-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 318-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 319-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 320-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 321-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 322-   Designed oligonucleotide primer for DNA sequencing-   SEQ ID NO: 323-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 324-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 325-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 326-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 327-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 328-   Designed oligonucleotide primer for PCR-   SEQ ID NO 329-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 330-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 331-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 332-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 333-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 334-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 335-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 336-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 337-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 338-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 339-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 340-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 341-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 342-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 343-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 344-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 345-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 346-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 347-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 348-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 349-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 350-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 351-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 352-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 353-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 354-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 355-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 356-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 357-   Designed oligonucleotide primer for PCR-   SEQ ID NO; 358-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 359-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 360-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 361-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 362-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 363-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 364-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 365-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 366-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 367-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 368-   Designed polynucleotide encoding amino acid sequence of SEQ ID    No.222-   SEQ ID NO: 369-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 370-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 371-   Designed oligonucleotide primer for PCR-   SEQ ID NO; 372-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 373-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 374-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 375-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 376-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 377-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 378-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 379-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 380-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 381-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 382-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 383-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 384-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 385-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 386-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 387-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 388-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 389-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 390-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 391-   Designed oligonucleotide primer for PCR-   SEQ DI NO: 392-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 393-   Designed polynucleotide encoding amino acid sequence of SEQ ID    No.224-   SEQ ID NO: 394-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 395-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 396-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 397-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 398-   Designed oligonucleotide primer for PCR-   SEQ ID NO: 399-   Designed oligonucleotide primer for PCR-   SEQ ID NO:400-   Designed oligonucleotide primer for PCR-   SEQ ID NO:401-   Designed oligonucleotide primer for PCR-   SEQ ID NO:402-   Designed oligonucleotide linker for construction of expression    vector-   SEQ ID NO:403-   Designed oligonucleotide linker for construction of expression    vector.

1. A DNA encoding a herbicide metabolizing protein, wherein said proteinis selected from the group consisting of: (A1) a protein comprising theamino acid sequence shown in SEQ ID NO: 1; (A2) a protein comprising theamino acid sequence shown in SEQ ID NO: 2; (A3) a protein comprising theamino acid sequence shown in SEQ ID NO: 3; (A4) a protein comprising theamino acid sequence shown in SEQ ID NO: 108; (A5) a protein having anability to convert in the presence of an electron transport systemcontaining an electron donor, a compound of formula (II):

to a compound of formula (III):

and comprising an amino acid sequence having at least 80% sequenceidentity with an amino acid sequence shown in any one of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 108; (A6) a protein having anability to convert in the presence of an electron transport systemcontaining an electron donor, a compound of formula (II) to a compoundof formula (III), and comprising an amino acid sequence encoded by anucleotide sequence having at least 80% sequence identity with anucleotide sequence encoding an amino acid sequence shown in any one ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 108; (A11) aprotein comprising the amino acid sequence shown in SEQ ID NO: 159;(A12) a protein comprising the amino acid sequence shown in SEQ ID NO:160; (A13) a protein comprising the amino acid sequence shown in SEQ IDNO: 136; (A14) a protein comprising the amino acid sequence shown in SEQID NO: 137; (A15) a protein comprising the amino acid sequence shown inSEQ ID NO: 138; (A16) a protein comprising the amino acid sequence shownin SEQ ID NO: 215; (A17) a protein comprising the amino acid sequenceshown in SEQ ID NO: 216; (A18) a protein comprising the amino acidsequence shown in SEQ ID NO: 217; (A19) a protein comprising the aminoacid sequence shown in SEQ ID NO: 218; (A20) a protein comprising theamino acid sequence shown in SEQ ID NO: 219; (A21) a protein comprisingthe amino acid sequence shown in SEQ ID NO: 220; (A22) a proteincomprising the amino acid sequence shown in SEQ ID NO: 221; (A23) aprotein comprising the amino acid sequence shown in SEQ ID NO: 222;(A24) a protein comprising the amino acid sequence shown in SEQ ID NO:223; (A25) a protein comprising the amino acid sequence shown in SEQ IDNO: 224; (A26) a protein having an ability to convert in the presence ofan electron transport system containing an electron donor, a compound offormula (II) to a compound of formula (III), and comprising an aminoacid sequence having at least 80% sequence identity with an amino acidsequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 136, SEQ ID NO:137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 220, SEQID NO: 221 or SEQ ID NO: 223 or an amino acid sequence having at least90% sequence identity with an amino acid sequence shown in any one ofSEQ ID NO: 160, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 1218, SEQ IDNO: 222 or SEQ ID NO: 224; A27) a protein having the ability to convertin the presence of an electron transport system containing an electrondonor, a compound of formula (II) to a compound of formula (III), andcomprising an amino acid sequence encoded by a nucleotide sequencehaving at least 90% sequence identity with a nucleotide sequenceencoding an amino acid sequence shown in any one of SEQ ID NO: 159, SEQID NO: 160, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ IDNO:215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO: 219,SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 or SEQ IDNO: 224; and (A28) a protein having an ability to convert in thepresence of an electron transport system containing an electron donor, acompound of formula (II) to a compound of formula (III), and comprisingan amino acid sequence encoded by a DNA amplifiable by a polymerasechain reaction with a primer comprising the nucleotide sequence shown inany one of SEQ ID NOs: 124 to 128, a primer comprising the nucleotidesequence shown in SEQ ID NO: 129 and as a template a chromosomal DNA ofStreptomyces phaeochromogenes, Streptomyces testaceus, Streptomycesachromogenes, Streptomyces griseofuscus, Streptomycesthermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis,Streptomyces glomerochromogenes, Streptomyces olivochromogenes,Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus,Streptomyces misawanensis, Streptomyces pallidus, Streptomycesroseorubens, Streptomyces rutgersensis, Streptomyces steffisburgensis orSaccharopolyspora taberi.
 2. A DNA comprising a nucleotide sequenceselected from the group consisting of: (a1) the nucleotide sequenceshown in SEQ ID NO: 6; (a2) the nucleotide sequence shown in SEQ ID NO:7; (a3) the nucleotide sequence shown in SEQ ID NO: 8; (a4) thenucleotide sequence shown in SEQ ID NO: 109; (a5) the nucleotidesequence shown in SEQ ID NO: 139; (a6) the nucleotide sequence shown inSEQ ID NO: 140; (a7) the nucleotide sequence shown in SEQ ID NO: 141;(a8) the nucleotide sequence shown in SEQ ID NO: 142; (a9) thenucleotide sequence shown in SEQ ID NO: 143; (a10) the nucleotidesequence shown in SEQ ID NO: 225; (a11) the nucleotide sequence shown inSEQ ID NO: 226; (a12) the nucleotide sequence shown in SEQ ID NO: 227;(a13) the nucleotide sequence shown in SEQ ID NO: 228; (a14) thenucleotide sequence shown in SEQ ID NO: 229; (a15) the nucleotidesequence shown in SEQ ID NO: 230; (a16) the nucleotide sequence shown inSEQ ID NO: 231 (a17) the nucleotide sequence shown in SEQ ID NO: 232;(a18) the nucleotide sequence shown in SEQ ID NO: 233; (a19) thenucleotide sequence shown in SEQ ID NO, 234; (a20) a nucleotide sequenceencoding an amino acid sequence of a protein having an ability toconvert in the presence of an electron transport system containing anelectron donor, a compound of formula (II) to a compound of formula(III), said nucleotide sequence having at least 80% sequence identitywith a nucleotide sequence shown in any one of SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 8 or SEQ ID: NO: 109; and (a21) a nucleotide sequenceencoding an amino acid sequence of a protein having an ability toconvert in the presence of an electron transport system containing anelectron donor, a compound of formula (II) to a compound of formula(III), said nucleotide sequence having at least 90% sequence identitywith a nucleotide sequence shown in any one of SEQ ID NO: 139, SEQ JDNO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 225,SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ IDNO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233 or SEQ ID NO:234.
 3. The DNA according to claim 1, comprising a nucleotide sequenceencoding an amino acid sequence of said protein, wherein the codon usagein said nucleotide sequence is within the range of plus or minus 4% ofthe codon usage in genes from the species of a host cell to which theDNA is introduced and the GC content of said nucleotide sequence is atleast 40% and at most 60%.
 4. A DNA comprising the nucleotide sequenceshown in SEQ ID NO:
 214. 5. A DNA comprising the nucleotide sequenceshown in SEQ ID NO:
 368. 6. A DNA comprising the nucleotide sequenceshown in SEQ ID NO:
 393. 7. A DNA in which a DNA having a nucleotidesequence encoding an intracellular organelle transit signal sequence islinked upstream of the DNA according to claim 1 in frame.
 8. A DNA inwhich the DNA according to claim 1 and a promoter functional in a hostcell are operably linked.
 9. A vector comprising the DNA according toclaim
 1. 10. A method of producing a vector comprising a step ofinserting the DNA according to claim 1 into a vector replicable in ahost cell.
 11. A transformant in which the DNA according to claim 1 isintroduced into a host cell.
 12. The transformant according to claim 11,wherein the host cell is a microorganism cell or a plant cell.
 13. Amethod of producing a transformant comprising a step of introducing intoa host cell, the DNA according to claim
 1. 14. A method of producing aprotein having the ability to convert a compound of formula (II) to acompound of formula (III), said method comprising a steps of culturingthe transformant according to claim 11 and recovering the produced saidprotein.
 15. Use of the DNA according to claim 1 for producing a proteinhaving the ability to convert a compound of formula (II) to a compoundof formula (III).
 16. A method of giving a plant resistance to aherbicide, said method comprising step of introducing into andexpressing in a plant cell, the DNA according to claim
 1. 17. Apolynucleotide having a partial nucleotide sequence of a DNA accordingto claim 1 or a nucleotide sequence complimentary to said partialnucleotide sequence.
 18. A method of detecting a DNA encoding a proteinhaving the ability to convert a compound of formula (II) to a compoundof formula (III), said method comprising a step of detecting a DNA towhich a probe is hybridized in a hybridization using as the probe theDNA according to claim 1 or the polynucleotide according to claim 17.19. A method of detecting a DNA encoding a protein having the ability toconvert a compound of formula (II) to a compound of formula (III), saidmethod comprising a step of detecting a DNA amplified in a polymerasechain reaction with the polynucleotide according to claim 17 as aprimer.
 20. The method according to claim 19, wherein at least one ofthe primers is selected from the group consisting of a polynucleotidecomprising the nucleotide sequence shown in any one of SEQ ID NOs: 124to 128 and a polynucleotide comprising the nucleotide sequence shown inSEQ ID NO:
 129. 21. A method of obtaining a DNA encoding a proteinhaving the ability to convert a compound of formula (II) to a compoundof formula (III), said method comprising a step of recovering the DNAdetected by the method according to claim 18 or
 19. 22. A method ofscreening a cell having a DNA encoding a protein having the ability toconvert a compound of formula (II) to a compound of formula (III), saidmethod comprising a step of detecting said DNA from a test cell by themethod according to claim 18 or
 19. 23. A herbicide metabolizing proteinselected from the group consisting of: (A1) a protein comprising theamino acid sequence shown in SEQ ID NO: 1; (A2) a protein comprising theamino acid sequence shown in SEQ ID NO: 2; (A3) a protein comprising theamino acid sequence shown in SEQ ID NO: 3; (A4) a protein comprising theamino acid sequence shown in SEQ ID NO: 108; (A5) a protein having anability to convert in the presence of an electron transport systemcontaining an electron donor, a compound of formula (II) to a compoundof formula (III) and comprising an amino acid sequence having at least80% sequence identity with an amino acid sequence shown in any one ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 108; (A6) aprotein having an ability to convert in the presence of an electrontransport system containing an electron donor, a compound of formula(II) to a compound of formula (III), and comprising an amino acidsequence encoded by a nucleotide sequence having at least 80% sequenceidentity with a nucleotide sequence encoding an amino acid sequenceshown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3 or SEQ IDNO: 108; (A11) a protein comprising the amino acid sequence shown in SEQID NO: 159; (A12) a protein comprising the amino acid sequence shown inSEQ ID NO: 160; (A13) a protein comprising the amino acid sequence shownin SEQ ID NO: 136; (A14) a protein comprising the amino acid sequenceshown in SEQ ID NO: 137; (A15) a protein comprising the amino acidsequence shown in SEQ ID NO: 138; (A16) a protein comprising the aminoacid sequence shown in SEQ ID NO: 215; (A17) a protein comprising theamino acid sequence shown in SEQ ID NO: 216; (A18) a protein comprisingthe amino acid sequence shown in SEQ ID NO: 217; (A19) a proteincomprising the amino acid sequence shown in SEQ ID NO: 218; (A20) aprotein comprising the amino acid sequence shown in SEQ ID NO: 219;(A21) a protein comprising the amino acid sequence shown in SEQ ID NO:220; (A22) a protein comprising the amino acid sequence shown in SEQ IDNO: 221; (A23) a protein comprising the amino acid sequence shown in SEQID NO: 222; (A24) a protein comprising the amino acid sequence shown inSEQ ID NO: 223; (A25) a protein comprising the amino acid sequence shownin SEQ ID NO: 224; (A26) a protein having an ability to convert in thepresence of an electron transport system containing an electron donor, acompound of formula (II) to a compound of formula (III), and comprisingan amino acid sequence having at least 80% sequence identity with anamino acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 136,SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219, SEQ IDNO: 220, SEQ ID NO: 221 or SEQ ID NO: 223 or an amino acid sequencehaving at least 90% sequence identity with an amino acid sequence shownin any one of SEQ ID NO: 160, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO:218, SEQ ID NO: 222 or SEQ ID NO: 224; (A27) a protein having theability to convert in the presence of an electron transport systemcontaining an electron donor, a compound of formula (II) to a compoundof formula (III), and comprising an amino acid sequence encoded by anucleotide sequence having at least 90% sequence identity with anucleotide sequence encoding an amino acid sequence shown in any one ofSEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 136, SEQ ID NO: 137, SEQ IDNO: 138, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218,SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ IDNO: 223 or SEQ ID NO: 224; and (A28) a protein having an ability toconvert in the presence of an electron transport system containing anelectron donor, a compound of formula (II) to a compound of formula(III), and comprising an amino acid sequence encoded by a DNAamplifiable by a polymerase chain reaction with a primer comprising thenucleotide sequence shown in any one of SEQ ID NOs: 124 to 128, a primercomprising the nucleotide sequence shown in SEQ ID NO: 129 and as atemplate a chromosomal DNA of Streptomyces phaeochromogenes,Streptomyces testaceus, Streptomyces achromogenes, Streptomycesgriseofuscus, Streptomyces thermocoerulescens, Streptomyces nogalater,Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomycesolivochromogenes, Streptomyces ornatus, Streptomyces griseus,Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus,Streptomyces roseorubens, Streptomyces rutgersensis, Streptomycessteffisburgensis or Saccharopolyspora taberi.
 24. An antibodyrecognizing a herbicide metabolizing protein selected from the groupconsisting of: (A1) a protein comprising the amino acid sequence shownin SEQ ID NO: 1, (A2) a protein comprising the amino acid sequence shownin SEQ ID NO: 2; (A3) a protein comprising the amino acid sequence shownin SEQ ID NO: 3; (A4) a protein comprising the amino acid sequence shownin SEQ ID NO: 108; (A5) a protein having an ability to convert in thepresence of an electron transport system containing an electron donor, acompound of formula (II) to a compound of formula (III) and comprisingan amino acid sequence having at least 80% sequence identity with anamino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3 or SEQ ID NO: 108; (A6) a protein having an ability to convertin the presence of an electron transport system containing an electrondonor, a compound of formula (II) to a compound of formula (III), andcomprising an amino acid sequence encoded by a nucleotide sequencehaving at least 80% sequence identity with a nucleotide sequenceencoding an amino acid sequence shown in any one of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3 or SEQ ID NO: 108; (A11) a protein comprising theamino acid sequence shown in SEQ ID NO: 159; (A12) a protein comprisingthe amino acid sequence shown in SEQ ID NO: 160; (A13) a proteincomprising the amino acid sequence shown in SEQ ID NO: 136; (A14) aprotein comprising the amino acid sequence shown in SEQ ID NO: 137;(A15) a protein comprising the amino acid sequence shown in SEQ ID NO:138; (A16) a protein comprising the amino acid sequence shown in SEQ IDNO: 215; (A17) a protein comprising the amino acid sequence shown in SEQID NO: 216; (A18) a protein comprising the amino acid sequence shown inSEQ ID NO: 217; (A19) a protein comprising the amino acid sequence shownin SEQ ID NO: 218; (A20) a protein comprising the amino acid sequenceshown in SEQ ID NO: 219; (A21) a protein comprising the amino acidsequence shown in SEQ ID NO: 220; (A22) a protein comprising the aminoacid sequence shown in SEQ ID NO: 221; (A23) a protein comprising theamino acid sequence shown in SEQ ID NO: 222; (A24) a protein comprisingthe amino acid sequence shown in SEQ ID NO: 223; (A25) a proteincomprising the amino acid sequence shown in SEQ ID NO: 224; (A26) aprotein having an ability to convert in the presence of an electrontransport system containing an electron donor, a compound of formula(II) to a compound of formula (III), and comprising an amino acidsequence having at least 80% sequence identity with an amino acidsequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 136, SEQ ID NO:137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 220, SEQID NO: 221 or SEQ ID NO: 223 or an amino acid sequence having at least90% sequence identity with an amino acid sequence shown in any one ofSEQ ID NO: 160, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 218, SEQ IDNO: 222 or SEQ ID NO: 224; (A27) a protein having the ability to convertin the presence of an electron transport system containing an electrondonor, a compound of formula (II) to a compound of formula (III), andcomprising an amino acid sequence encoded by a nucleotide sequencehaving at least 90% sequence identity with a nucleotide sequenceencoding an amino acid sequence shown in any one of SEQ ID NO: 159, SEQID NO: 160, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO:215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO: 219, SEQID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 or SEQ ID NO:224; and (A28) a protein having an ability to convert in the presence ofan electron transport system containing an electron donor, a compound offormula (II) to a compound of formula (III), and comprising an aminoacid sequence encoded by a DNA amplifiable by a polymerase chainreaction with a primer comprising the nucleotide sequence shown in anyone of SEQ ID NOs: 124 to 128, a primer comprising the nucleotidesequence shown in SEQ ID NO: 129 and as a template a chromosomal DNA ofStreptomyces phaeochromogenes, Streptomyces testaceus, Streptomycesachromogenes, Streptomyces griseofuscus, Streptomycesthermocoerulescens, Streptomyces nogalater, Streptomyces tsusimaensis,Streptomyces glomerochromogenes, Streptomyces olivochromogenes,Streptomyces ornatus, Streptomyces griseus, Streptomyces lanatus,Streptomyces misawanensis, Streptomyces pallidus, Streptomycesroseorubens, Streptomyces rutgersensis, Streptomyces steffisburgensis orSaccharopolyspora taberi.
 25. A method of detecting a herbicidemetabolizing protein, said method comprising: (3) a step of contacting atest substance with an antibody recognizing said protein and (4) a stepof detecting a complex of said protein and said antibody, arising fromsaid contact, wherein said protein is selected from the group consistingof: (A1) a protein comprising the amino acid sequence shown in SEQ IDNO: 1; (A2) a protein comprising the amino acid sequence shown in SEQ IDNO: 2; (A3) a protein comprising the amino acid sequence shown in SEQ IDNO: 3; (A4) a protein comprising the amino acid sequence shown in SEQ IDNO: 108; (A5) a protein having an ability to convert in the presence ofan electron transport system containing an electron donor, a compound offormula (II) to a compound of formula (III) and comprising an amino acidsequence having at least 80% sequence identity with an amino acidsequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 orSEQ ID NO: 108; (A6) a protein having an ability to convert in thepresence of an electron transport system containing an electron donor, acompound of formula (II) to a compound of formula (III), and comprisingan amino acid sequence encoded by a nucleotide sequence having at least80% sequence identity with a nucleotide sequence encoding an amino acidsequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 orSEQ ID NO: 108; (A11) a protein comprising the amino acid sequence shownin SEQ ID NO: 159; (A12) a protein comprising the amino acid sequenceshown in SEQ ID NO: 160; (A13) a protein comprising the amino acidsequence shown in SEQ ID NO: 136; (A14) a protein comprising the aminoacid sequence shown in SEQ ID NO: 137; (A15) a protein comprising theamino acid sequence shown in SEQ ID NO: 138; (A16) a protein comprisingthe amino acid sequence shown in SEQ ID NO: 215; (A17) a proteincomprising the amino acid sequence shown in SEQ ID NO: 216; (A18) aprotein comprising the amino acid sequence shown in SEQ ID NO: 217;(A19) a protein comprising the amino acid sequence shown in SEQ ID NO:218; (A20) a protein comprising the amino acid sequence shown in SEQ IDNO: 219; (A21) a protein comprising the amino acid sequence shown in SEQID NO: 220; (A22) a protein comprising the amino acid sequence shown inSEQ ID NO: 221; (A23) a protein comprising the amino acid sequence shownin SEQ ID NO: 222; (A24) a protein comprising the amino acid sequenceshown in SEQ ID NO: 223; (A25) a protein comprising the amino acidsequence shown in SEQ ID NO: 224; (A26) a protein having an ability toconvert in the presence of an electron transport system containing anelectron donor, a compound of formula (II) to a compound of formula(III), and comprising an amino acid sequence having at least 80%sequence identity with an amino acid sequence shown in any one of SEQ IDNO: 159, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 217,SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221 or SEQ ID NO: 223 or anamino acid sequence having at least 90% sequence identity with an aminoacid sequence shown in any one of SEQ ID NO: 160, SEQ ID NO: 215, SEQ IDNO: 216, SEQ ID NO: 218, SEQ ID NO: 222 or SEQ ID NO: 224; A27) aprotein having the ability to convert in the presence of an electrontransport system containing an electron donor, a compound of formula(II) to a compound of formula (III), and comprising an amino acidsequence encoded by a nucleotide sequence having at least 90% sequenceidentity with a nucleotide sequence encoding an amino acid sequenceshown in any one of SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 136, SEQID NO: 137, SEQ ID NO: 138, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQID NO: 222, SEQ ID NO: 223 or SEQ ID NO: 224; and (A28) a protein havingan ability to convert in the presence of an electron transport systemcontaining an electron donor, a compound of formula (II) to a compoundof formula (III), and comprising an amino acid sequence encoded by a DNAamplifiable by a polymerase chain reaction with a primer comprising thenucleotide sequence shown in any one of SEQ ID NOs: 124 to 128, a primercomprising the nucleotide sequence shown in SEQ ID NO: 129 and as atemplate a chromosomal DNA of Streptomyces phaeochromogenes,Streptomyces testaceus, Streptomyces achromogenes, Streptomycesgriseofuscus, Streptomyces thermocoerulescens, Streptomyces nogalater,Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomycesolivochromogenes, Streptomyces ornatus, Streptomyces griseus,Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus,Streptomyces roseorubens, Streptomyces rutgersensis, Streptomycessteffisburgensis or Saccharopolyspora taberi.
 26. An analysis ordetection kit comprising the antibody according to claim
 24. 27. A DNAencoding a ferredoxin selected from the group consisting of: (B1) aprotein comprising an amino acid sequence shown in SEQ ID NO: 12; (B2) aprotein comprising an amino acid sequence shown in SEQ ID NO: 13; (B3) aprotein comprising an amino acid sequence shown in SEQ ID NO: 14; (B4) aprotein comprising an amino acid sequence shown in SEQ ID NO: 111; (B5)a ferredoxin comprising an amino acid sequence having at least 80%sequence identity with an amino acid sequence shown in any one of SEQ IDNO: 12, SEQ ID NO: 13, SEQ ID NO 14 or SEQ ID NO: 111; (B6) a ferredoxincomprising an amino acid sequence encoded by a nucleotide sequencehaving at least 90% sequence identity with a nucleotide sequenceencoding an amino acid sequence shown in any one of SEQ ID NO: 12, SEQID NO: 13, SEQ ID NO 14 or SEQ ID NO: 111; (B7) a protein comprising anamino acid sequence shown in SEQ ID NO: 149; (B8) a protein comprisingan amino acid sequence shown in SEQ ID NO: 150; (B9) a proteincomprising an amino acid sequence shown in SEQ ID NO: 151; (B10) aprotein comprising an amino acid sequence shown in SEQ ID NO: 152; (B11)a protein comprising an amino acid sequence shown in SEQ ID NO: 153;(B12) a ferredoxin comprising an amino acid sequence having at least 80%sequence identity with an amino acid sequence shown in any one of SEQ IDNO: 149, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 245,SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ IDNO: 251, or SEQ ID NO: 253 or an amino acid sequence having at least 90%sequence identity with an amino acid sequence shown in any one of SEQ IDNO: 150, SEQ ID NO: 252 or SEQ ID NO: 254; (B13) a ferredoxin comprisingan amino acid sequence encoded by a nucleotide sequence having at least90% sequence identity with a nucleotide sequence encoding an amino acidsequence shown in any one of SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO:151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 245, SEQ ID NO: 247, SEQID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO:252, SEQ ID NO: 253 or SEQ ID NO: 254; (B14) a protein comprising theamino acid sequence shown in SEQ ID NO: 245; (B15) a protein comprisingthe amino acid sequence shown in SEQ ID NO: 247; (B16) a proteincomprising the amino acid sequence shown in SEQ ID NO: 248; (B17) aprotein comprising the amino acid sequence shown in SEQ ID NO: 249;(B18) a protein comprising the amino acid sequence shown in SEQ ID NO:250; (B19) a protein comprising the amino acid sequence shown in SEQ IDNO: 251; (B20) a protein comprising the amino acid sequence shown in SEQID NO: 252; (B21) a protein comprising the amino acid sequence shown inSEQ ID NO: 253; and (B22) a protein comprising the amino acid sequenceshown in SEQ ID NO:
 254. 28. A DNA comprising a nucleotide sequenceselected from the group consisting of: (b1) a nucleotide sequence shownin SEQ ID NO: 15; (b2) a nucleotide sequence shown in SEQ ID NO: 16;(b3) a nucleotide sequence shown in SEQ ID NO: 17; (b4) a nucleotidesequence shown in SEQ ID NO: 112; (b5) a nucleotide sequence shown inSEQ ID NO: 154; (b6) a nucleotide sequence shown in SEQ ID NO: 155; (b7)a nucleotide sequence shown in SEQ ID NO: 156; (b8) a nucleotidesequence shown in SEQ ID NO: 157; (b9) a nucleotide sequence shown inSEQ ID NO: 158; (b10) a nucleotide sequence shown in SEQ ID NO: 255;(b11) a nucleotide sequence shown in SEQ ID NO: 257; (b12) a nucleotidesequence shown in SEQ ID NO: 258; (b13) a nucleotide sequence shown inSEQ ID NO: 259; (b14) a nucleotide sequence shown in SEQ ID NO: 260;(b15) a nucleotide sequence shown in SEQ ID NO: 261; (16) a nucleotidesequence shown in SEQ ID NO: 262; (b17) a nucleotide sequence shown inSEQ ID NO: 263; (b18) a nucleotide sequence shown in SEQ ID NO: 264; and(b19) a nucleotide sequence having at least 90% sequence identity with anucleotide sequence shown in any one of SEQ ID NO: 15, SEQ ID NO: 16,SEQ ID NO: 17, SEQ ID NO: 112, SEQ ID NO: 154, SEQ ID NO: 155, SEQ IDNO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 255, SEQ ID NO: 257,SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ IDNO: 262, SEQ ID NO: 263 or SEQ ID NO:
 264. 29. A vector comprising a DNAaccording to claim
 28. 30. A transformant in which the DNA according toclaim 28 is introduced into a st cell.
 31. A ferredoxin selected fromthe group consisting of: (B1) a protein comprising an amino acidsequence shown in SEQ ID NO: 12; (B2) a protein comprising an amino acidsequence shown in SEQ ID NO: 13; (B3) a protein comprising an amino acidsequence shown in SEQ ID NO: 14; (B4) a protein comprising an amino acidsequence shown in SEQ ID NO: 111; (B5) a ferredoxin comprising an aminoacid sequence having at least 80% sequence identity with an amino acidsequence shown in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14or SEQ ID NO: 111; (B6) a ferredoxin comprising an amino acid sequenceencoded by a nucleotide sequence having at least 90% sequence identitywith a nucleotide sequence encoding an amino acid sequence shown in anyone of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO 14 or SEQ ID NO: 111;(B7) a protein comprising an amino acid sequence shown in SEQ ID NO:149; (B8) a protein comprising an amino acid sequence shown in SEQ IDNO: 150; (B9) a protein comprising an amino acid sequence shown in SEQID NO: 151; (B10) a protein comprising an amino acid sequence shown inSEQ ID NO: 152; (B11) a protein comprising an amino acid sequence shownin SEQ ID NO: 153; (B12) a ferredoxin comprising an amino acid sequencehaving at least 80% sequence identity with an amino acid sequence shownin any one of SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO:153, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQID NO: 250, SEQ ID NO: 251, or SEQ ID NO: 253 or an amino acid sequencehaving at least 90% sequence identity with an amino acid sequence shownin any one of SEQ ID NO: 150, SEQ ID NO: 252, or SEQ ID NO: 254; (B13) aferredoxin comprising an amino acid sequence encoded by a nucleotidesequence having at least 90% sequence identity with a nucleotidesequence encoding an amino acid sequence shown in any one of SEQ ID NO:149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQID NO: 245, SEQ ID NO: 247, SEQ ID NO; 248, SEQ ID NO: 249, SEQ ID NO:250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253 or SEQ ID NO: 254;(B14) a protein comprising the amino acid sequence shown in SEQ ID NO:245; (B15) a protein comprising the amino acid sequence shown in SEQ IDNO: 247; (B16) a protein comprising the amino acid sequence shown in SEQID NO: 248; (B17) a protein comprising the amino acid sequence shown inSEQ ID NO: 249; (B18) a protein comprising the amino acid sequence shownin SEQ ID NO: 250; (B19) a protein comprising the amino acid sequenceshown in SEQ ID NO: 251; (820) a protein comprising the amino acidsequence shown in SEQ ID NO: 252; (B21) a protein comprising the aminoacid sequence shown in SEQ ID NO: 253; and (B22) a protein comprisingthe amino acid sequence shown in SEQ ID NO:
 254. 32. A DNA comprising anucleotide sequence selected from the group consisting of: (ab1) anucleotide sequence shown in SEQ ID NO: 9; (ab2) a nucleotide sequenceshown in SEQ ID NO: 10; (ab3) a nucleotide sequence shown in SEQ ID NO:11; (ab4) a nucleotide sequence shown in SEQ ID NO: 110; (ab5) anucleotide sequence shown in SEQ ID NO: 144; (ab6) a nucleotide sequenceshown in SEQ ID NO: 145; (ab7) a nucleotide sequence shown in SEQ ID NO:146; (ab8) a nucleotide sequence shown in SEQ ID NO, 147; (ab9) anucleotide sequence shown in SEQ ID NO: 148; (ab10) a nucleotidesequence shown in SEQ ID NO: 235; (ab11) a nucleotide sequence shown inSEQ ID NO: 236; (ab12) a nucleotide sequence shown in SEQ ID NO: 237;(ab13) a nucleotide sequence shown in SEQ ID NO: 238; (ab14) anucleotide sequence shown in SEQ ID NO: 239; (ab15) a nucleotidesequence shown in SEQ ID NO: 240; (ab16) a nucleotide sequence shown inSEQ ID NO: 241; (ab17) a nucleotide sequence shown in SEQ ID NO: 242;(ab18) a nucleotide sequence shown in SEQ ID NO: 243; and (ab19) anucleotide sequence shown in SEQ ID NO:
 244. 33. A vector comprising theDNA according to claim
 32. 34. A transformant in which the DNA accordingto claim 32 is introduced into a host cell.
 35. The transformantaccording to claim 34, wherein the host cell is a microorganism cell ora plant cell.
 36. A method of producing a transformant comprising a stepof introducing into a host cell the DNA according to claim
 32. 37. Amethod of producing a protein having the ability to convert a compoundof formula (II) to a compound of formula (III), said method comprising astep of culturing the transformant according to claim 34 and recoveringthe produced said protein.
 38. A method of controlling weeds comprisinga step of applying a compound to a cultivation area of a plantexpressing at least one herbicide metabolizing protein selected from thegroup consisting of: (A1) a protein comprising the amino acid sequenceshown in SEQ ID NO: 1; (A2) a protein comprising the amino acid sequenceshown in SEQ ID NO: 2; (A3) a protein comprising the amino acid sequenceshown in SEQ ID NO: 3; (A4) a protein comprising the amino acid sequenceshown in SEQ ID NO: 108; (A5) a protein having an ability to convert inthe presence of an electron transport system containing an electrondonor, a compound of formula (II) to a compound of formula (III), andcomprising an amino acid sequence having at least 80% sequence identitywith an amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 108; (A6) a protein having an ability toconvert in the presence of an electron transport system containing anelectron donor, a compound of formula (II) to a compound of formula(III), and comprising an amino acid sequence encoded by a nucleotidesequence having at least 80% sequence identity with a nucleotidesequence encoding an amino acid sequence shown in any one of SEQ ID NO;1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 108; (A7) a protein havingthe ability to convert in the presence of an electron transport systemcontaining an electron donor, a compound of formula (II) to a compoundof formula (III), and comprising an amino acid sequence encoded by a DNAthat hybridizes, under stringent conditions, to a DNA comprising anucleotide sequence encoding an amino acid sequence shown in any one ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 108; (A8) aprotein having the ability to convert in the presence of an electrontransport system containing an electron donor, a compound of formula(II) to a compound of formula (III), and comprising an amino acidsequence encoded by a DNA amplifiable by a polymerase chain reactionwith a primer comprising a nucleotide sequence shown in SEQ ID NO: 129,a primer comprising a nucleotide sequence shown in any one of SEQ IDNOs: 124 to 128, and as a template a chromosome of a microorganismbelonging to Streptomyces or Saccharopolyspora; (A9) a proteincomprising an amino acid sequence shown in SEQ ID NO: 4; (A11) a proteincomprising the amino acid sequence shown in SEQ ID NO: 159; (A12) aprotein comprising the amino acid sequence shown in SEQ ID NO: 160;(A13) a protein comprising the amino acid sequence shown in SEQ ID NO:136; (A14) a protein comprising the amino acid sequence shown in SEQ IDNO: 137; (A15) a protein comprising the amino acid sequence shown in SEQID NO: 138; (A16) a protein comprising the amino acid sequence shown inSEQ ID NO: 215; (A17) a protein comprising the amino acid sequence shownin SEQ ID NO: 216; (A18) a protein comprising the amino acid sequenceshown in SEQ ID NO: 217; (A19) a protein comprising the amino acidsequence shown in SEQ ID NO: 218; (A20) a protein comprising the aminoacid sequence shown in SEQ ID NO: 219; (A21) a protein comprising theamino acid sequence shown in SEQ ID NO: 220; (A22) a protein comprisingthe amino acid sequence shown in SEQ ID NO: 221; (A23) a proteincomprising the amino acid sequence shown in SEQ ID NO: 222; (A24) aprotein comprising the amino acid sequence shown in SEQ ID NO: 223;(A25) a protein comprising the amino acid sequence shown in SEQ ID NO:224; (A26) a protein having an ability to convert in The presence of anelectron transport system containing an electron donor, a compound offormula (II) to a compound of formula (III), and comprising an aminoacid sequence having at least 80% sequence identity with an amino acidsequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 136, SEQ ID NO:137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 220, SEQID NO: 221 or SEQ ID NO: 223 or an amino acid sequence having at least90% sequence identity with an amino acid sequence shown in any one ofSEQ ID NO: 160, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 218, SEQ IDNO 222 or SEQ ID NO: 224; and (A27) a protein having the ability toconvert in the presence of an electron transport system containing anelectron donor, a compound of formula (II) to a compound of formula(III), and comprising an amino acid sequence encoded by a nucleotidesequence having at least 90% sequence identity with a nucleotidesequence encoding an amino acid sequence shown in any one of SEQ ID NO:159, SEQ ID NO: 160, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 orSEQ ID NO: 224, wherein said compound is a compound of formula (I):

wherein in formula (I) G represents a group shown in any one of thefollowing G-1 to G-9:

wherein in G-1 to G-9, X represents an oxygen atom or sulfur atom; Yrepresents an oxygen atom or sulfur atom; R¹ represents a hydrogen atomor halogen atom; R² represents a hydrogen atom, C₁-C₈ alkyl group, C₁-C₈haloalkyl group, halogen atom, hydroxyl group, —OR⁹ group, —SH group,—S(O)pR⁹ group, —COR⁹ group, —CO₂R⁹ group, —C(O)SR⁹ group, —C(O)NR¹¹R¹²group, —CONH₂ group, —CHO group, —CR⁹═NOR¹⁸ group, —CH═CR¹⁹CO₂R⁹ group,—CH₂CHR¹⁹CO₂R⁹ group, —CO₂N═CR¹³R¹⁴ group, nitro group, cyano group,—NHSO₂R¹⁵ group, —NHSO₂NHR⁵ group, —NR⁹R²⁰ group, —NH₂ group or phenylgroup that may be substituted with one or more C₁-C₄ alkyl groups whichmay be the same or different; p represents 0, 1 or 2; R³ representsC₁-C₂ alkyl group, C₁-C₂ haloalkyl group, —OCH₃ group, —SCH₃ group,—OCHF₂ group, halogen atom, cyano group, nitro group or C₁-C₃ alkoxygroup substituted with a phenyl group which may be substituted on thering with at least one substituent selected from a halogen atom, C₁-C₃alkyl group, C₁-C₃ haloalkyl group, OR²⁸ group, NR¹¹R²⁸ group, SR²⁸group, cyano group, CO₂R²⁸ group and nitro group; R⁴ represents ahydrogen atom, C₁-C₃ alkyl group or C₁-C₃ haloalkyl group; R⁵ representsa hydrogen atom, C₁-C₃ alkyl group, C₁-C₃ haloalkyl group, cyclopropylgroup, vinyl group, C₂ alkynyl group, cyano group, —C(O)R²⁰ group,—CO₂R²⁰ group, —C(O)NR²⁰R²¹ group, —CHR¹⁶R¹⁷CN group, —CR¹⁶R¹⁷C(O)R²⁰group, —C¹⁶R¹⁷CO₂R²⁰ group, —CR¹⁶R¹⁷C(O)NR²⁰R²¹ group, CHR¹⁶0H group,—CHR¹⁶OC(O)R²⁰ group or —OCHR¹⁶C(O)NR²⁰R²¹ group, or, when G representsG-2 or G-6, R⁴ and R⁵ may represent C═O group together with the carbonatom to which they are attached; R⁶ represents C₁-C₆ alkyl group, C₁-C₆haloalkyl group, C₂-C₆ alkoxyalkyl group, C₃-C₆alkenyl group or C₃-C₆alkynyl group; R⁷ represents a hydrogen atom, C₁-C₆ alkyl group, C₁-C₆haloalkyl group, halogen atom, —S(O)₂(C₁-C₆ alkyl) group or —C(═O)R²²group; R⁸ represents a hydrogen atom, C₁-C₈ alkyl group, C₃-C₈cycloalkyl group, C₃-C₈ alkenyl group, C₃-C₈ alkynyl group, C₁-C₉haloalkyl group, C₂-C₈ alkoxyalkyl group, C₃-C₈ alkoxyalkoxyalkyl group,C₃-C₈ haloalkynyl group, C₃-C₈ haloalkenyl group, C₁-C₈ alkylsulfonylgroup, C₁-C₈ haloalkylsulfonyl group, C₃-C₈ alkoxycarbonylalkyl group,—S(O)₂NH(C₁-C₈ alkyl) group, —C(O)R²³ group or benzyl group which may besubstituted with R²⁴ on the phenyl ring; R⁹ represents C₁-C₈ alkylgroup, C₃-C₈ cycloalkyl group, C₃-C₈ alkenyl group, C₃-C₈ alkynyl group,C₁-C₈ haloalkyl group, C₂-C₈ alkoxyalkyl group, C₂-C₈ alkylthioalkylgroup, C₂-C₈ alkylsulfinylalkyl group, C₂-C₈ alkylsulfonylalkyl group,C₄-C₈ alkoxyalkoxyalkyl group, C₄-C₈ cycloalkylalkyl group, C₄-C₈cycloalkoxyalkyl group, C₄-C₈ alkenyloxyalkyl group, C₄-C₈alkynyloxyalkyl group, C₃-C₈ haloalkoxyalkyl group, C₄-C₈haloalkenyloxyalkyl group, C₄-C₈ haloalkynyloxyalkyl group, C₄-C₈cycloalkylthioalkyl group, C₄-C₈ alkenylthioalkyl group, C₄-C₈alkynylthioalkyl group, C₁-C₄ alkyl group substituted with a phenoxygroup which may be substituted on the ring with at least one substituentselected from a halogen atom, C₁-C₃ alkyl group and C₁-C₃ haloalkylgroup, C₁-C₄ allyl group substituted with a benzyloxy group which may besubstituted on the ring with at least one substituent selected from ahalogen atom, C₁-C₃ alkyl group and C₁-C₃ haloalkyl group, C₄-C₈trialkylsyrylalkyl group, C₂-C₈ cyanoalkyl group, C₃-C₈ halocycloalkylgroup, C₃-C₈ haloalkenyl group, C₅-C₈ alkoxyalkenyl group, C₅-C₈haloalkoxyalkenyl group, C₅-C₅ alkylthioalkenyl group, C₅-C₈ haloalkynylgroup, C₅-C₈ alkoxyalkynyl group, C₅-C₈ haloalkoxyalkynyl group, C₅-C₈alkylthioalkynyl group, C₂-C₈ alkylcarbonyl group, benzyl group whichmay be substituted on the ring with at least one substituent selectedfrom a halogen atom, C₁-C₃ alkyl group, C₁-C₃ haloalkyl group, OR²⁸group, —NR¹¹R²⁸ group, —SR²⁸ group, cyano group, —CO₂R²⁸ group and nitrogroup, —CR¹⁶R¹⁷ COR¹⁰ group, —CR¹⁶R¹⁷CO₂R²⁰ group, —CR¹⁶R¹⁷P(O)(OR¹⁰)₂group, —CR¹⁶R¹⁷P(S)(OR¹⁰)₂ group, —CR¹⁶R¹⁷C(O)NR¹¹R¹² group,—CR¹⁶R¹⁷C(O)NH₂ group, —C(═CR²R²⁷)COR¹⁰ group, —C(═CR²⁶R²⁷)CO₂R²⁰ group,—C(═CR²⁶R²⁷)P(O)(OR¹⁰)₂ group, —C(═CR²⁶R²⁷)P(S)(OR¹⁰)₂ group,—C(═CR²⁶R²⁷)C(O)NR¹¹R¹² group, —C(═CR²⁶R²⁷)C(O)NH₂ group, or any one ofrings shown in Q-1 to Q-7:

which may be substituted on the ring with at least one substituentselected from a halogen atom, C₁-C₆ alkyl group, C₁-C₆ haloalkyl group,C₂-C₆ alkenyl group, C₂-C₆ haloalkenyl group, C₂-C₆ alkynyl group, C₃-C₆haloalkynyl group, C₂-C₈ alkoxyalkyl group, R²⁸ group, —SR²⁸ group,—NR¹¹R²⁸ group, C₃-C₈ alkoxycarbonylalkyl group, C₂-C₄ carboxyalkylgroup, —CO₂R²⁸ group and cyano group; R¹⁰ represents a C₁-C₆ alkylgroup, C₂-C₆ alkenyl group, C₃-C₆ alkynyl group or tetrahydrofuranylgroup; R¹¹ and R¹³ independently represent a hydrogen atom or C₁-C₄allyl group; R¹² represents C₁-C₆ alkyl group, C₃-C₆ cycloalkyl group,C₃-C₆ alkenyl group, C₃-C₆ alkynyl group, C₂-C₆ alkoxyalkyl group, C₁-C₆haloalkyl group, C₃-C₆ haloalkenyl group, C₃-C₆ haloalkynyl group,phenyl group which may be substituted on the ring with at least onesubstituent selected from a halogen atom, C₁-C₄ alkyl group and C₁-C₄alkoxy group or —CR¹⁶R¹⁷CO₂R²⁵ group; or, R¹¹ and R¹² together mayrepresent —(CH₂)₅—, —(CH₂)₄— or —CH₂CH₂OCH₂CH₂—, or in that case theresulting ring may be substituted with a substituent selected from aC₁-C₃ alkyl group, a phenyl group and benzyl group; R¹⁴ represents aC₁-C₄ alkyl group or phenyl group which may be substituted on the ringwith a substituent selected from a halogen atom, C₁-C₃ alkyl group andC₁-C₃ haloalkyl group; or, R¹³ and R¹⁴ may represent C₃-C₈ cycloalkylgroup together with the carbon atom to which they are attached; R¹⁵represents C₁-C₄ allyl group, C₁-C₄ haloalkyl group or C₃-C₆ alkenylgroup; R¹⁶ and R¹⁷ independently represent a hydrogen atom or C₁-C₄alkyl group, C₁-C₄ haloalkyl group, C₂-C₄ alkenyl group, C₂-C₄haloalkenyl group, C₂-C₄ alkynyl group, C₃-C₄ haloalkynyl group; or, R¹⁶and R¹⁷ may represent C₃-C₆ cycloalkyl group with the carbon atom towhich they are attached, or the ring thus formed may be substituted withat least one substituent selected from a halogen atom, a C₁-C₃ alkylgroup and C₁-C₃ haloalkyl group; R¹⁸ represents a hydrogen atom, C₁-C₆alkyl group, C₃-C₆ alkenyl group or C₃-C₆ alkynyl group; R¹⁹ representsa hydrogen atom, C₁-C₄ alkyl group or halogen atom, R²⁰ represents ahydrogen atom, C₁-C₆ alkyl group, C₃-C₆ cycloalkyl group, C₃-C₆ alkenylgroup, C₃-C₆ alkynyl group, C₂-C₆ alkoxyalkyl group, C₁-C₆ haloalkylgroup, C₃-C₆ haloalkenyl group, C₃-C₆ haloalkynyl group, phenyl groupwhich may be substituted on the ring with at least one substituentselected from a halogen atom, C₁-C₄ alkyl group and —OR²⁸ group, or—CR¹⁶R¹⁷CO₂R²⁵ group; R²¹ represents a hydrogen atom, C₁-C₂ alkyl groupor —CO₂(C₁-C₄ alkyl) group; R²² represents a hydrogen atom, C₁-C₆ alkylgroup, C₁-C₆ alkoxy group or NH(C₁-C₆ alkyl) group; R²³ represents C₁-C₆alkyl group, C₁-C₆ haloalkyl group, C₁-C₆ alkoxy group, NH(C₁-C₆ alkyl)group, benzyl group, C₂-C₈ dialkylamino group or phenyl group which maybe substituted with R²⁴; R²⁴ represents C₁-C₆ alkyl group, 1 to 2halogen atoms, C₁-C₆ alkoxy group or CF₃ group; R²⁵ represents C₁-C₆alkyl group, C₁-C₆ haloalkyl group, C₃-C₆ alkenyl group, C₃-C₆haloalkenyl group, C₃-C₆ alkynyl group or C₃-C₆ haloalkynyl group; R²⁶and R²⁷ each represent independently a hydrogen atom, C₁-C₄ alkyl group,C₁-C₄ haloalkyl group, C₂-C₄ alkenyl group, C₂-C₄ haloalkenyl group,C₂-C₄ alkynyl group, C₃-C₄ haloalkynyl group, —OR²⁸ group, —NHR²⁸ group,or —SR²⁸ group; or, R²⁶ and R²⁷ may represent C₃-C₈ cycloalkyl groupwith the carbon atom to which they are attached, or each of the ringthus formed may be substituted with at least one substituent selectedfrom a halogen atom, C₁-C₃ alkyl group and C₁-C₃ haloalkyl group; and,R²⁸ represents a hydrogen atom, C₁-C₆ alkyl group, C₁-C₆ haloalkylgroup, C₃-C₆ alkenyl group, C₃-C₆ haloalkenyl group, C₃-C₆ alkynylgroup, C₃-C₆ haloalkynyl group, C₂-C₄ carboxyalkyl group, C₃-C₈alkoxycarbonylalkyl group, C₃-C₈ haloalkoxycarbonylalkyl group, C₅-C₉alkenyloxycabonylalkyl group, C₅-C₉ haloalkenyloxycabonylalkyl group,C₅-C₉ alkynyloxycabonylalkyl group, C₅-C₉ haloalkynyloxycabonylalkylgroup, C₅-C₉ cycloalkoxycabonylalkyl group or C₅-C₉halocycloalkoxycabonylalkyl group.
 39. A method of controlling weedscomprising a step of applying a compound to a cultivation area of aplant expressing at least one protein selected from the group consistingof: (A1) a protein comprising the amino acid sequence shown in SEQ IDNO: 1; (A2) a protein comprising the amino acid sequence shown in SEQ IDNO: 2, (A3) a protein comprising the amino acid sequence shown in SEQ IDNO: 3; (A4) a protein comprising the amino acid sequence shown in SEQ IDNO: 108; (A5) a protein having an ability to convert in the presence ofan electron transport system containing an electron donor, a compound offormula (II) to a compound of formula (III) and comprising an amino acidsequence having at least 80% sequence identity with an amino acidsequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 orSEQ ID NO: 108; (A6) a protein having an ability to convert in thepresence of an electron transport system containing an electron donor, acompound of formula (II) to a compound of formula (III), and comprisingan amino acid sequence encoded by a nucleotide sequence having at least80% sequence identity with a nucleotide sequence encoding an amino acidsequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO; 3 orSEQ ID NO: 108; (A11) a protein comprising the amino acid sequence shownin SEQ ID NO: 159; (A12) a protein comprising the amino acid sequenceshown in SEQ ID NO: 160; (A13) a protein comprising the amino acidsequence shown in SEQ ID NO: 136; (A14) a protein comprising the aminoacid sequence shown in SEQ ID NO: 137; (A15) a protein comprising theamino acid sequence shown in SEQ ID NO: 138; (A16) a protein comprisingthe amino acid sequence shown in SEQ ID NO: 215; (A17) a proteincomprising the amino acid sequence shown in SEQ ID NO: 216; (A18) aprotein comprising the amino acid sequence shown in SEQ ID NO: 217;(A19) a protein comprising the amino acid sequence shown in SEQ ID NO:218; (A20) a protein comprising the amino acid sequence shown in SEQ IDNO: 219; (A21) a protein comprising the amino acid sequence shown in SEQID NO: 220; (A22) a protein comprising the amino acid sequence shown inSEQ ID NO:221; (A23) a protein comprising the amino acid sequence shownin SEQ ID NO: 222; (A24) a protein comprising the amino acid sequenceshown in SEQ ID NO: 223; (A25) a protein comprising the amino acidsequence shown in SEQ ID NO: 224; (A26) a protein having an ability toconvert in the presence of an electron transport system containing anelectron donor, a compound of formula (II) to a compound of formula(III), and comprising an amino acid sequence having at least 80%sequence identity with an amino acid sequence shown in any one of SEQ IDNO: 159, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 217,SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221 or SEQ ID NO; 223 or anamino acid sequence having at least 90% sequence identity with an aminoacid sequence shown in any one of SEQ ID NO: 160, SEQ ID NO: 215, SEQ IDNO-216, SEQ ID NO: 218, SEQ ID NO: 222 or SEQ ID NO: 224; (A27) aprotein having the ability to convert in the presence of an electrontransport system containing an electron donor, a compound of formula(II) to a compound of formula (III), and comprising an amino acidsequence encoded by a nucleotide sequence having at least 90% sequenceidentity with a nucleotide sequence encoding an amino acid sequenceshown in any one of SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 136, SEQID NO: 137, SEQ ID NO-138, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO;217, SEQ ID NO:218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQID NO: 222, SEQ ID NO: 223 or SEQ ID NO: 224; and (A28) a protein havingan ability to convert in the presence of an electron transport systemcontaining an electron donor, a compound of formula (II) to a compoundof formula (III), and comprising an amino acid sequence encoded by a DNAamplifiable by a polymerase chain reaction with a primer comprising thenucleotide sequence shown in any one of SEQ ID NOs: 124 to 128, a primercomprising the nucleotide sequence shown in SEQ ID NO: 129 and as atemplate a chromosomal DNA of Streptomyces phaeochromogenes,Streptomyces testaceus, Streptomyces achromogenes, Streptomycesgriseofuscus, Streptomyces thermocoerulescens, Streptomyces nogalater,Streptomyces tsusimaensis, Streptomyces glomerochromogenes, Streptomycesolivochromogenes, Streptomyces ornatus, Streptomyces griseus,Streptomyces lanatus, Streptomyces misawanensis, Streptomyces pallidus,Streptomyces roseorubens, Streptomyces rutgersensis, Streptomycessteffisburgensis or Saccharopolyspora taberi.
 40. A method of evaluatingthe resistance of a cell to a compound of formula (I), said methodcomprising: (3) a step of contacting said compound with a cellexpressing at least one herbicide metabolizing protein selected from thegroup consisting of: (A1) a protein comprising the amino acid sequenceshown in SEQ ID NO: 1; (A2) a protein comprising the amino acid sequenceshown in SEQ ID NO: 2; (A3) a protein comprising the amino acid sequenceshown in SEQ ID NO: 3; (A4) a protein comprising the amino acid sequenceshown in SEQ ID NO: 108; (A5) a protein having an ability to convert inthe presence of an electron transport system containing an electrondonor, a compound of formula (II) to a compound of formula (III) andcomprising an amino acid sequence having at least 80% sequence identitywith an amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 108; (A6) a protein having an ability toconvert in the presence of an electron transport system containing anelectron donor, a compound of formula (II) to a compound of formula(III), and comprising an amino acid sequence encoded by a nucleotidesequence having at least 80% sequence identity with a nucleotidesequence encoding an amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 108; (A7) a protein havingthe ability to convert in the presence of an electron transport systemcontaining an electron donor, a compound of formula (II) to a compoundof formula (III), and comprising an amino acid sequence encoded by a DNAthat hybridizes, under stringent conditions, to a DNA comprising anucleotide sequence encoding an amino acid sequence shown in any one ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 108; (A8) aprotein having the ability to convert in the presence of an electrontransport system containing an electron donor, a compound of formula(II) to a compound of formula (III), and comprising an amino acidsequence encoded by a DNA amplifiable by a polymerase chain reactionwith a primer comprising a nucleotide sequence shown in SEQ ID NO: 129,a primer comprising a nucleotide sequence shown in any one of SEQ IDNOs: 124 to 128, and as a template a chromosome of a microorganismbelonging to Streptomyces or Saccharopolyspora; (A9) a proteincomprising an amino acid sequence shown in SEQ ID NO: 4; (A11) a proteincomprising the amino acid sequence shown in SEQ ID NO: 159; (A12) aprotein comprising the amino acid sequence shown in SEQ ID NO: 160;(A13) a protein comprising the amino acid sequence shown in SEQ ID NO:136; (A14) a protein comprising the amino acid sequence shown in SEQ IDNO: 137; (A15) a protein comprising the amino acid sequence shown in SEQID NO: 138; (A16) a protein comprising the amino acid sequence shown inSEQ ID NO: 215; (A17) a protein comprising the amino acid sequence shownin SEQ ID NO: 216; (A18) a protein comprising the amino acid sequenceshown in SEQ ID NO: 217; (A19) a protein comprising the amino acidsequence shown in SEQ ID NO: 218; (A20) a protein comprising the aminoacid sequence shown in SEQ ID NO: 219; (A21) a protein comprising theamino acid sequence shown in SEQ ID NO: 220; (A22) a protein comprisingthe amino acid sequence shown in SEQ ID NO: 221; (A23) a proteincomprising the amino acid sequence shown in SEQ ID NO: 222; (A24) aprotein comprising the amino acid sequence shown in SEQ ID NO: 223;(A25) a protein comprising the amino acid sequence shown in SEQ ID NO:224; (A26) a protein having an ability to convert in the presence of anelectron transport system containing an electron donor, a compound offormula (II) to a compound of formula (III), and comprising an aminoacid sequence having at least 80% sequence identity with an amino acidsequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 136, SEQ ID NO:137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 220, SEQID NO: 221 or SEQ ID NO: 223 or an amino acid sequence having at least90% sequence identity with an amino acid sequence shown in any one ofSEQ ID NO: 160, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 218, SEQ IDNO: 222 or SEQ ID NO: 224; and (A27) a protein having the ability toconvert in the presence of an electron transport system containing anelectron donor, a compound of formula (II) to a compound of formula(III), and comprising an amino acid sequence encoded by a nucleotidesequence having at least 90% sequence identity with a nucleotidesequence encoding an amino acid sequence shown in any one of SEQ ID NO:159, SEQ ID NO: 160, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 orSEQ ID NO: 224; and (4) a step of evaluating the degree of damage to thecell which contacted the compound in the above step (1).
 41. The methodaccording to claim 40, wherein the cell is a microorganism cell orplaint cell.
 42. A method of selecting a cell resistant to a compound offormula (1), said method comprising a step of selecting a cell based onthe resistance evaluated in the method according to claim
 40. 43. Thecell resistant to herbicide selected by the method according to claim42, or the culture thereof.
 44. A method of evaluating the resistance ofa plant to a compound of formula (1), said method comprising: (3) a stepof contacting said compound with a plant expressing at least oneherbicide metabolizing protein selected from the group consisting of:(A1) a protein comprising the amino acid sequence shown in SEQ ID NO: 1;(A2) a protein comprising the amino acid sequence shown in SEQ ID NO: 2;(A3) a protein comprising the amino acid sequence shown in SEQ ID NO: 3;(A4) a protein comprising the amino acid sequence shown in SEQ ID NO:108; (A5) a protein having an ability to convert in the presence of anelectron transport system containing an electron donor, a compound offormula (II) to a compound of formula (III) and comprising an amino acidsequence having at least 80% sequence identity with an amino acidsequences shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3or SEQ ID NO: 108; (A6) a protein having an ability to convert in thepresence of an electron transport system containing an electron donor, acompound of formula (II) to a compound of formula (III), and comprisingan amino acid sequence encoded by a nucleotide sequence having at least80% sequence identity with a nucleotide sequence encoding an amino acidsequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 orSEQ ID NO: 108; (A7) a protein having the ability to convert in thepresence of an electron transport system containing an electron donor, acompound of formula (II) to a compound of formula (III), and comprisingan amino acid sequence encoded by a DNA that hybridizes, under stringentconditions, to a DNA comprising a nucleotide sequence encoding an aminoacid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQID NO: 108; (A8) a protein having the ability to convert in the presenceof an electron transport system containing an electron donor a compoundof formula (II) to a compound of formula (III), and comprising an aminoacid sequence encoded by a DNA amplifiable by a polymerase chainreaction with a primer comprising a nucleotide sequence shown in SEQ IDNO: 129, a primer comprising a nucleotide sequence shown in any one ofSEQ ID NOs: 124 to 128, and as a template a chromosome of amicroorganism belonging to Streptomyces or Saccharopolyspora; (A9) aprotein comprising an amino acid sequence shown in SEQ ID NO: 4; (A11) aprotein comprising the amino acid sequence shown in SEQ ID NO: 159;(A12) a protein comprising the amino acid sequence shown in SEQ ID NO:160; (A13) a protein comprising the amino acid sequence shown in SEQ IDNO: 136; (A14) a protein comprising the amino acid sequence shown in SEQID NO: 137; (A15) a protein comprising the amino acid sequence shown inSEQ ID NO; 138; (A16) a protein comprising the amino acid sequence shownin SEQ ID NO: 215; (A17) a protein comprising the amino acid sequenceshown in SEQ ID NO: 216; (A18) a protein comprising the amino acidsequence shown in SEQ ID NO: 217; (A19) a protein comprising the aminoacid sequence shown in SEQ ID NO: 218; (A20) a protein comprising theamino acid sequence shown in SEQ ID NO: 219; (A21) a protein comprisingthe amino acid sequence shown in SEQ ID NO: 220; (A22) a proteincomprising the amino acid sequence shown in SEQ ID NO: 221; (A23) aprotein comprising the amino acid sequence shown in SEQ ID NO: 222;(A24) a protein comprising the amino acid sequence shown in SEQ ID NO:223; (A25) a protein comprising the amino acid sequence shown in SEQ IDNO: 224; (A26) a protein having an ability to convert in the presence ofan electron transport system containing an electron donor, a compound offormula (II) to a compound of formula (III), and comprising an aminoacid sequence having at least 80% sequence identity with an amino acidsequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 136, SEQ ID NO;137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 220, SEQID NO: 221 or SEQ ID NO: 223 or an amino acid sequence having at least90% sequence identity with an amino acid sequence shown in any one ofSEQ ID NO: 160, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 218, SEQ IDNO: 222 or SEQ ID NO: 224; and (A27) a protein having the ability toconvert in the presence of an electron transport system containing anelectron donor a compound of formula (II) to a compound of formula(III), and comprising an amino acid sequence encoded by a nucleotidesequence having at least 90% sequence identity with a nucleotidesequence encoding an amino acid sequence shown in any one of SEQ ID NO:159, SEQ ID NO: 160, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 orSEQ ID NO: 224; and (4) a step of evaluating the degree of damage to theplant which contacted the compound described in step (1).
 45. A methodof selecting a plant resistant to a compound of formula (I), said methodcomprising a step of selecting a plant based on the resistance evaluatedin the method according to claim
 44. 46. A herbicidally resistant plantselected from the method according to claim 45, or the progeny thereof.47. A method of treating a compound of formula (1), said methodcomprising reacting said compound in the presence of an electrontransport system containing an electron donor, with at least oneherbicide metabolizing protein selected from the group consisting of:(A1) a protein comprising the amino acid sequence shown in SEQ ID NO: 1;(A2) a protein comprising the amino acid sequence shown in SEQ ID NO: 2;(A3) a protein comprising the amino acid sequence shown in SEQ ID NO: 3;(A4) a protein comprising the amino acid sequence shown in SEQ ID NO:108; (A5) a protein having an ability to convert in the presence of anelectron transport system containing an electron donor, a compound offormula (II) to a compound of formula (III) and comprising an amino acidsequence having at least 80% sequence identity with an amino acidsequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 orSEQ ID NO: 108; (A6) a protein having an ability to convert in thepresence of an electron transport system containing an electron donor, acompound of formula (II) to a compound of formula (III), and comprisingan amino acid sequence encoded by a nucleotide sequence having at least80% sequence identity with a nucleotide sequence encoding an amino acidsequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 orSEQ ID NO: 108; (A7) a protein having the ability to convert in thepresence of an electron transport system containing an electron donor, acompound of formula (II) to a compound of formula (III), and comprisingan amino acid sequence encoded by a DNA that hybridizes, under stringentconditions, to a DNA comprising a nucleotide sequence encoding an aminoacid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3 or SEQ ID NO: 108; (A8) a protein having the ability to convert in thepresence of an electron transport system containing an electron donor, acompound of formula (II) to a compound of formula (III), and comprisingan amino acid sequence encoded by a DNA amplifiable by a polymerasechain reaction with a primer comprising a nucleotide sequence shown inSEQ ID NO: 129, a primer comprising a nucleotide sequence shown in anyone of SEQ ID NOs: 124 to 128, and as a template a chromosome of amicroorganism belonging to Streptomyces or Saccharopolyspora; (A9) aprotein comprising an amino acid sequence shown in SEQ ID NO: 4; (A11) aprotein comprising the amino acid sequence shown in SEQ ID NO: 159;(A12) a protein comprising the amino acid sequence shown in SEQ ID NO:160; (A13) a protein comprising the amino acid sequence shown in SEQ IDNO: 136; (A14) a protein comprising the amino acid sequence shown in SEQID NO: 137; (A15) a protein comprising the amino acid sequence shown inSEQ ID NO: 138; (A16) a protein comprising the amino acid sequence shownin SEQ ID NO: 215; (A17) a protein comprising the amino acid sequenceshown in SEQ ID NO: 216; (A18) a protein comprising the amino acidsequence shown in SEQ ID NO: 217; (A19) a protein comprising the aminoacid sequence shown in SEQ ID NO: 218; (A20) a protein comprising theamino acid sequence shown in SEQ ID NO: 219; (A21) a protein comprisingthe amino acid sequence shown in SEQ ID NO: 220; (A22) a proteincomprising the amino acid sequence shown in SEQ ID NO: 221; (A23) aprotein comprising the amino acid sequence shown in SEQ ID NO: 222;(A24) a protein comprising the amino acid sequence shown in SEQ ID NO:223; (A25) a protein comprising the amino acid sequence shown in SEQ IDNO: 224; (A26) a protein having an ability to convert in the presence ofan electron transport system containing an electron donor a compound offormula (II) to a compound of formula (III), and comprising an aminoacid sequence having at least 80% sequence identity with an amino acidsequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 136, SEQ ID NO:137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 220, SEQID NO: 221 or SEQ ID NO: 223 or an amino acid sequence having at least90% sequence identity with an amino acid sequence shown in any one ofSEQ ID NO: 160, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 218, SEQ IDNO: 222 or SEQ ID NO, 224; and (A27) a protein having the ability toconvert in the presence of an electron transport system containing anelectron donor, a compound of formula (II) to a compound of formula(III), and comprising an amino acid sequence encoded by a nucleotidesequence having at least 90% sequence identity with a nucleotidesequence encoding an amino acid sequence shown in any one of SEQ ID NO:159, SEQ ID NO: 160, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 orSEQ ID NO:
 224. 48. The method according to claim 47, wherein reactingthe compound with the herbicide metabolizing protein by contacting thecompound with a transformant in which a DNA encoding the herbicidemetabolizing protein is introduced into a host cell in a positionenabling its expression in said cell.
 49. Use for treating the compoundof formula (I) of a herbicide metabolizing protein selected from thegroup consisting of: (A1) a protein comprising the amino acid sequenceshown in SEQ ID NO: 1; (A2) a protein comprising the amino acid sequenceshown in SEQ ID NO: 2; (A3) a protein comprising the amino acid sequenceshown in SEQ ID NO: 3; (A4) a protein comprising the amino acid sequenceshown in SEQ ID NO: 108; (A5) a protein having an ability to convert inthe presence of an electron transport system containing an electrondonor, a compound of formula (II) to a compound of formula (III) andcomprising an amino acid sequence having at least 80% sequence identitywith an amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 108; (A6) a protein having an ability toconvert in the presence of an electron transport system containing anelectron donor, a compound of formula (II) to a compound of formula(III), and comprising an amino acid sequence encoded by a nucleotidesequence having at least 80% sequence identity with a nucleotidesequence encoding any one of the amino acid sequences shown in any oneof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 108; (A7) aprotein having the ability to convert in the presence of an electrontransport system containing an electron donor, a compound of formula(II) to a compound of formula (III), and comprising an amino acidsequence encoded by a DNA that hybridizes, under stringent conditions,to a DNA comprising a nucleotide sequence encoding an amino acidsequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:108; (A8) a protein having the ability to convert in the presence of anelectron transport system containing an electron donor, a compound offormula (II) to a compound of formula (III), and comprising an aminoacid sequence encoded by a DNA amplifiable by a polymerase chainreaction with a primer comprising a nucleotide sequence shown in SEQ IDNO: 129, a primer comprising a nucleotide sequence shown in any one ofSEQ ID NOs: 124 to 128, and as a template chromosome of a microorganismbelonging to Streptomyces or Saccharopolyspora; (A9) a proteincomprising an amino acid sequence shown in SEQ ID NO: 4; (A11) a proteincomprising the amino acid sequence shown in SEQ ID NO: 159; (A12) aprotein comprising the amino acid sequence shown in SEQ ID NO: 160;(A13) a protein comprising the amino acid sequence shown in SEQ ID NO:136; (A14) a protein comprising the amino acid sequence shown in SEQ IDNO: 137; (A15) a protein comprising the amino acid sequence shown in SEQID NO: 138; (A16) a protein comprising the amino acid sequence shown inSEQ ID NO: 215; (A17) a protein comprising the amino acid sequence shownin SEQ ID NO: 216; (A18) a protein comprising the amino acid sequenceshown in SEQ ID NO: 217; (A19) a protein comprising the amino acidsequence shown in SEQ ID NO: 218; (A20) a protein comprising the aminoacid sequence shown in SEQ ID NO: 219; (A21) a protein comprising theamino acid sequence shown in SEQ ID NO: 220; (A22) a protein comprisingthe amino acid sequence shown in SEQ ID NO: 221; (A23) a proteincomprising the amino acid sequence shown in SEQ ID NO: 222; (A24) aprotein comprising the amino acid sequence shown in SEQ ID NO: 223;(A25) a protein comprising the amino acid sequence shown in SEQ ID NO:224; (A26) a protein having an ability to convert in the presence of anelectron transport system containing an electron donor, a compound offormula (II) to a compound of formula (III), and comprising an aminoacid sequence having at least 80% sequence identity with an amino acidsequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 136, SEQ ID NO:137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 220, SEQID NO: 221 or SEQ ID NO: 223 or an amino acid sequence having at least90% sequence identity with an amino acid sequence shown in any one ofSEQ ID NO: 160, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 218, SEQ IDNO: 222 or SEQ ID NO: 224; and (A27) a protein having the ability toconvert in the presence of an electron transport system containing anelectron donor, a compound of formula (II) to a compound of formula(III), and comprising an amino acid sequence encoded by a nucleotidesequence having at least 90% sequence identity with a nucleotidesequence encoding the amino acid sequence shown in any one of SEQ ID NO:159, SEQ ID NO: 160, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223 orSEQ ID NO:
 224. 50. Use for treating a compound of formula (I) of apolynucleotide encoding a herbicide metabolizing protein selected fromthe group consisting of (A1) a protein comprising the amino acidsequence shown in SEQ ID NO: 1; (A2) a protein comprising the amino acidsequence shown in SEQ ID NO: 2; (A3) a protein comprising the amino acidsequence shown in SEQ ID NO: 3; (A4) a protein comprising the amino acidsequence shown in SEQ ID NO: 108; (A5) a protein having an ability toconvert in the presence of an electron transport system containing anelectron donor, a compound of formula (II) to a compound of formula(III) and comprising an amino acid sequence having at least 80% sequenceidentity with an amino acid sequence shown in any one of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 108; (A6) a protein having anability to convert in the presence of an electron transport systemcontaining an electron donor a compound of formula (II) to a compound offormula (III), and comprising an amino acid sequence encoded by anucleotide sequence having at least 80% sequence identity with anucleotide sequence encoding an amino acid sequence shown in any one ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 108; (A7) aprotein having the ability to convert in the presence of an electrontransport system containing an electron donor, a compound of formula(II) to a compound of formula (III), and comprising an amino acidsequence encoded by a DNA that hybridizes, under stringent conditions,to a DNA comprising a nucleotide sequence encoding an amino acidsequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:108; (A8) a protein having the ability to convert in the presence of anelectron transport system containing an electron donor, a compound offormula (II) to a compound of formula (III), and comprising an aminoacid sequence encoded by a DNA amplifiable by a polymerase chainreaction with a primer comprising a nucleotide sequence shown in SEQ IDNO: 129, a primer comprising a nucleotide sequence shown in any one ofSEQ ID NOs: 124 to 128, and as a template a chromosome of amicroorganism belonging to Streptomyces or Saccharopolyspora; (A9) aprotein comprising an amino acid sequence shown in SEQ ID NO: 4; (A11) aprotein comprising the amino acid sequence shown in SEQ ID NO: 159;(A12) a protein comprising the amino acid sequence shown in SEQ ID NO:160; (A13) a protein comprising the amino acid sequence shown in SEQ IDNO: 136; (A14) a protein comprising the amino acid sequence shown in SEQID NO; 137; (A15) a protein comprising the amino acid sequence shown inSEQ ID NO: 138; (A16) a protein comprising the amino acid sequence shownin SEQ ID NO: 215; (A17) a protein comprising the amino acid sequenceshown in SEQ ID NO: 216; (A18) a protein comprising the amino acidsequence shown in SEQ ID NO: 217; (A19) a protein comprising the aminoacid sequence shown in SEQ ID NO: 218; (A20) a protein comprising theamino acid sequence shown in SEQ ID NO: 219; (A21) a protein comprisingthe amino acid sequence shown in SEQ ID NO; 220; (A22) a proteincomprising the amino acid sequence shown in SEQ ID NO: 221; (A23) aprotein comprising the amino acid sequence shown in SEQ ID NO: 222;(A24) a protein comprising the amino acid sequence shown in SEQ ID NO:223; (A25) a protein comprising the amino acid sequence shown in SEQ IDNO: 224; (A26) a protein comprising an ability to convert in thepresence of an electron transport system containing an electron donor, acompound of formula (II) to a compound of formula (III), and comprisingan amino acid sequence having at least 80% sequence identity with anamino acid sequence shown in any one of SEQ ID NO: 159, SEQ ID NO: 136,SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 217, SEQ ID NO: 219, SEQ IDNO: 220, SEQ ID NO: 221 or SEQ ID NO: 223 or an amino acid sequencehaving at least 90% sequence identity with an amino acid sequence shownin any one of SEQ ID NO: 160, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO:218, SEQ ID NO: 222 or SEQ ID NO: 224; and (A27) a protein having theability to convert in the presence of an electron transport systemcontaining an electron donor, a compound of formula (II) to a compoundof formula (III), and comprising an amino acid sequence encoded by anucleotide sequence having at least 90% sequence identity with anucleotide sequence encoding an amino acid sequence shown in any one ofSEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 136, SEQ ID NO: 137, SEQ IDNO: 138, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218,SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO; 221, SEQ ID NO: 222, SEQ IDNO: 223 or SEQ ID NO: 224.